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Biparental care is a critical and, occasionally, unequally shared obligation that ensures that young survive to maturity. Such care may be complicated in systems in which one parent, typically the male, is unsure of his genetic relatedness to the young. Males may reduce paternal provisioning when full paternity is not assured, as occurs in mating systems in which females engage in extrapair copulations. Moreover, other factors independent of extrapair matings, such as male personality traits, likely also affect the level of paternal care. In this study, we determined the effect of a paternity threat event (i.e., a conspecific or a heterospecific territory intrusion) and male personality (i.e., the level of aggressiveness) on provisioning effort by male house wrens (Troglodytes aedon). Males were more likely to attack a conspecific intruder than a heterospecific intruder. Males that were exposed to a conspecific intruder were less likely to provision young at all. Of those males that did feed the young in their nest, male aggressiveness did not relate to feeding effort. These findings suggest that the likelihood of paternal care is reduced by perceived threats to paternity but that the costs of not feeding potentially multisired young are high and feeding efforts are unrelated to male personality.

Differences in avian eggshell pigmentation could be an honest signal of female quality that males use to inform their nestling provisioning effort. We investigated whether among-individual variation in protoporphyrin-based eggshell pigmentation in house wrens (Troglodytes aedon) reflects female fitness-associated traits and whether males use that information. Females laying lighter clutches were older and larger than females laying darker clutches. Nestlings hatching from lighter clutches had greater size-corrected mass on post-hatch day 11, a measure that strongly predicts survival and recruitment to the breeding population. To test whether male provisioning effort responds to clutch pigmentation, we used a reciprocal clutch cross-fostering design, swapping dark with light clutches and light with dark; in controls, we swapped light with light clutches and dark with dark. Shortly before hatching, clutches were returned to their original nest to avoid confounding effects of nestling quality on male provisioning. Contrary to the sexual selection hypothesis, clutch pigmentation had no effect on male provisioning. Males were probably able to observe eggshell pigmentation and thus had information about female quality, but they did not use this information to modulate their nestling provisioning. This may be because of constraints on species-specific reproductive opportunities, or because variation in eggshell protoporphyrin serves other functions.

Diseases caused by various Mycobacterium sp., especially Mycobacterium tuberculosis, are a major burden on global health care. Due to high intrinsic antibiotic resistance, treatment options are severely limited. In mycobacteria, WhiB7 coordinates intrinsic resistance to a broad range of antibiotics. While WhiB7 has been established as an auto-regulatory transcriptional activator, the signals and genes needed to induce its expression are poorly understood. Using Mycobacterium smegmatis as a model, we coupled transposon mutagenesis and next generation sequencing with WhiB7-specific antibiotic selection to identify genes that contribute to WhiB7 regulation and function. We showed that whiB7 expression was regulated by two coordinated processes: early termination of the whiB7 transcript and increased whiB7 promoter activity. Early termination was irreversibly maintained by constitutive expression of a putative aspartate aminotransferase gene, MSMEG_4060. A pair of hypothetical genes, MSMEG_3637 and MSMEG_3638, were identified as important contributors to whiB7 promoter induction on antibiotic challenge. Expansion of our understanding of the WhiB7-resistance pathway may lead to identification of inhibitors that allow the use of previously ineffective antibiotics to treat mycobacterial diseases.

Variation in parental age can have important consequences for offspring fitness and the structure of populations and disease transmission. However, our understanding of the effects of parental age on offspring in natural populations is limited. Here, we investigate consequences of parental age for offspring fitness and test for age-assortative mating in a short-lived bird, the house wren (Troglodytes aedon). Offspring immunoresponsiveness increased with maternal age and decreased with paternal age, but the strength of these effects varied with the age of one’s mate. Offspring immunoresponsiveness was augmented most with older mothers and younger fathers. Thus, we expected this combination of ages to yield the highest offspring fitness. However, offspring recruitment, longevity, and lifetime reproductive success were greatest when both parents were of above-average age. Consistent with the interactive effects of parental age on offspring fitness, we detected positive age-assortative mating among breeding pairs. Our results suggest that selection favors age-assortative mating, but in different ways depending on how parental ages affect offspring. We suggest that, in this short-lived species, selection for combinations of parental ages that maximize offspring immune responses is likely weaker than selection to produce breeding adults.

Infections caused by nontuberculous mycobacteria (NTM) are increasing worldwide, resulting in a new global health concern. NTM treatment is complex and requires combinations of several drugs for lengthy periods. In spite of this, NTM disease is often associated with poor treatment outcomes. The anti-parasitic family of macrocyclic lactones (ML) (divided in two subfamilies: avermectins and milbemycins) was previously described as having activity against mycobacteria, including Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium marinum, among others. Here, we aimed to characterize the in vitro anti-mycobacterial activity of ML against a wide range of NTM species, including Mycobacteroides abscessus. For this, Minimum Inhibitory Concentration (MIC) values of eight ML were determined against 80 strains belonging to nine different NTM species. Macrocyclic lactones showed variable ranges of anti-mycobacterial activity that were compound and species-dependent. Milbemycin oxime was the most active compound, displaying broad-spectrum activity with MIC lower than 8 mg/L. Time kill assays confirmed MIC data and showed bactericidal and sterilizing activity of some compounds. Macrocyclic lactones are available in many formulations and have been extensively used in veterinary and human medicine with suitable pharmacokinetics and safety properties. This information could be exploited to explore repurposing of anti-helminthics for NTM therapy.

The injection of phytohaemagglutinin (PHA) and sampling of blood are widely used in studies of wild vertebrates to assess components of immune and endocrine function and health state and to obtain genetic material. Despite the pervasive use of these techniques in the life sciences, their potential effects on survival are rarely considered. For example, whether injection of the immunogen PHA into body parts critical for locomotion (e.g., the prepatagium, or wing web, in birds) affects survival has not been tested. Here, we test whether injection of PHA into the wing web and blood sampling from nestling house wrens affects their subsequent recruitment and survival as breeding adults. Capture‐mark‐recapture analysis on a large sample of young (N = 20,152 fledglings from 3959 broods) treated over 10 years revealed that neither PHA injection nor blood sampling affected individual survival and detection probability. Recruitment as a breeder varied among years, but this variation was not attributable to sampling effort, or the percent of all adults identified at the nest during a given year. Variation in the percent of adults identified was primarily attributable to the effect of nest depredation on our ability to capture nesting pairs. Our results indicating lack of an effect of blood sampling and immune stimulation on survival are encouraging, but we recommend further work to assess the potential negative effects of all commonly used techniques on the survival of study subjects in the wild, including the potential costs associated with mounting various immunological responses.

Mycobacterium abscessus is a rapidly emerging mycobacterial pathogen causing dangerous pulmonary infections. Because these bacteria are intrinsically multidrug resistant, treatment options are limited and have questionable efficacy. The current treatment regimen relies on a combination of antibiotics, including clarithromycin paired with amikacin and either imipenem or cefoxitin. Tigecycline may be added when triple therapy is ineffective. We initially screened a library containing the majority of clinically available antibiotics for anti- M. abscessus activity. The screen identified rifabutin, which was then investigated for its interactions with M. abscessus antibiotics used in drug regimens. Combination of rifabutin with either clarithromycin or tigecycline generated synergistic anti- M. abscessus activity, dropping the rifabutin MIC below concentrations found in the lung. Importantly, these combinations generated bactericidal activity. The triple combination of clarithromycin, tigecycline, and rifabutin was also synergistic, and clinically relevant concentrations had a sterilizing effect on M. abscessus cultures. We suggest that combinations including rifabutin should be further investigated for treatment of M. abscessus pulmonary infections.

Parental and territory quality are often correlated in territorial birds, and both factors influence the resources allocated to offspring. Surprisingly, the relative contribution of these two components of variation in parental investment remains obscure. We experimentally decoupled the normal covariation between parental quality and territory quality to test the hypothesis that territory quality influences female prenatal and postnatal reproductive allocation. Territories were categorized into low-, intermediate-, and high-quality based on fledging success of nests over the previous 6 years (nesting sites are fixed in space).To decouple covariation between territory quality and individual quality, nestbox entrance size was increased on high-quality territories and left small on poor-quality sites because house wrens (Troglodytes aedon) prefer small over large entrances to their nest sites. We found a significant prenatal effect of territory quality on nestling provisioning: when reared on intermediate-quality territories, nestlings hatching from eggs produced on low-quality territories were provisioned at a higher rate than those hatching from eggs produced on high-quality territories. We propose that the increased provisioning was brought about by increased nestling begging mediated by a maternally derived compound, such as corticosterone, transferred to the eggs of stressed females in poor-quality habitat.

Cholesterol catabolism is thought to be a key factor contributing to the pathogenesis of Mycobacterium tuberculosis. Previous epistasis and mutant screening studies predicted that the P55 efflux pump (Rv1410c) positively interacts with the Mce4 transporter, a major cholesterol import system of M. tuberculosis and is needed for optimal growth in vitro, in macrophages, and in vivo. Using a combination of cell growth kinetic techniques, cholesterol consumption, and [4-(14)C]cholesterol uptake studies, we demonstrated that the Mycobacterium bovis BCG rv1410c gene indeed is needed for optimal in vitro growth on cholesterol and other carbon sources. Our data, together with previous predictions, support hypotheses that the P55 efflux pump functions in maintaining general metabolism or as a subunit of the Mce4 transport apparatus (catalyzing its assembly or providing cell wall integrity) to allow more efficient cholesterol uptake.

The incidence of concussions and potential for long-term health effects has captured the attention of the media, general public, medical professionals, parents, and obviously the athletes themselves. Concussions have been blamed for a variety of mental and physical health issues. The athletic trainer is at the forefront of the concussion management team, as they are typically on the scene when the concussion occurs and are often the first medical personnel to evaluate and, hopefully, remove the athlete from activity. There has been controversy of late regarding the influence of coaches in the care of concussed athletes. Therefore, a move to the “medical model” of sports medicine management can go a long way in resolving conflict of interest issues regarding the care of concussed athletes. A comprehensive concussion team and protocol are also essential to providing the highest level of care. This article takes a closer look at concussion management in the collegiate arena, with a particular focus on Princeton University.

Combinations of antibiotics, each individually effective against Mycobacterium abscessus , are routinely coadministered based on the concept that this minimizes the spread of antibiotic resistance. However, our in vitro data contradict this assumption and instead document antagonistic interactions between two antibiotics (clarithromycin and amikacin) used to treat M. abscessus infections. Clinically relevant concentrations of clarithromycin induced increased resistance to both amikacin and itself. The induction of resistance was dependent on whiB7 , a transcriptional activator of intrinsic antibiotic resistance that is induced by exposure to many different antibiotics. In M. abscessus , the deletion of whiB7 (MAB_3508c) resulted in increased sensitivity to a broad range of antibiotics. WhiB7 was required for transcriptional activation of genes that confer resistance to three commonly used anti- M. abscessus drugs: clarithromycin, amikacin, and tigecycline. The whiB7 -dependent gene that conferred macrolide resistance was identified as erm (41) (MAB_2297), which encodes a ribosomal methyltransferase. The whiB7 -dependent gene contributing to amikacin resistance was eis2 (MAB_4532c), which encodes a Gcn5-related N -acetyltransferase (GNAT). Transcription of whiB7 and the resistance genes in its regulon was inducible by subinhibitory concentrations of clarithromycin but not by amikacin. Thus, exposure to clarithromycin, or likely any whiB7 -inducing antibiotic, may antagonize the activities of amikacin and other drugs. This has important implications for the management of M. abscessus infections, both in cystic fibrosis (CF) and non-CF patients.

In biparental species, conflict between the sexes is expected when optimal levels of parental care differ between males and females. We studied food provisioning in house wrens (Troglodytes aedon) to test whether male provisioning is more strongly affected than female provisioning by brood size, because this should influence the marginal return on a male’s investment. To test this, we created broods of reduced and enlarged size, and found that per-nestling food delivery varied more strongly for males than for females in association with brood size and nestling age; males delivered more food per nestling to enlarged broods, and less to small broods, as nestlings grew and approached asymptotic size. Although parents delivered a greater amount of prey to larger broods, the amount of food per nestling was initially higher for nestlings in small broods, and the amount of food delivered early in nestling development, particularly by males, affected offspring body mass, survival to fledging, and recruitment to the breeding population. However, provisioning rates were negatively associated with prey size, suggesting a trade-off between prey quantity and size. Finally, parents provisioning at a high rate to broods of reduced size were less likely to return to breed, suggesting that they assessed the low return on their investment and dispersed. Although potential for sexual conflict in house wrens remains, our data suggest that its resolution does not involve negotiation over provisioning, possibly because selection for offspring viability favors heavy investment by both parents.

Aggressive signals should predict whether the sender of the signal will attack the receiver, yet this criterion has been little studied. We conducted experiments with male House Wrens (Troglodytes aedon )i n north- central Illinois in 2009 to test the hypothesis that rates of song delivery and wing-quivering (putative aggressive signals) signal aggressive intent. We simulated a conspecific territorial intrusion by combining playback of male song with a male taxidermic mount, predicting that these signals would be related to a male's likelihood of attacking a conspecific. All males (N = 37) sang in response to the intrusion. Males attacking the mount sang at significantly higher rates and performed significantly more wing quivers than males that did not attack. In addition, all males that attacked the mount performed wing quivers (9/9), whereas only 53.6% (15/28) of males that did not attack did so (P = 0.011). Our results are consistent with the hypothesis that both song and wing quivering indicate a signaler's aggressive intent and that these signals are likely components of a multi-component, hierarchical display. RESUMEN. Los despliegues agresivos de la Ratona Com´ un estcompuestos por m´ ultiples componentes que predicen el ataque Las se˜ nales de agresividad deber´ oan predecir si el emisor de la se˜ nal atacaral receptor, a´ un as´ o este criterio ha sido poco estudiado. Realizamos experimentos en la Ratona Com´ (Troglodytes aedon) en el centro norte de Illinois en 2009 a fin de poner a prueba la hip´ otesis de que la tasa de emisi´ on del canto y de temblor de alas (se˜ nales putativas de agresividad) se˜ nalizan la intenci´ nales probablemente sean componentes de un despliegue jery de m´ ultiples componentes.

Beak abnormalities have been reported in a wide range of species but typically affect only a small portion (

Theory predicts that a mother’s ability to produce high-quality offspring should influence whether she produces sons or daughters. We tested this hypothesis in house wrens (Troglodytes aedon) using a within-clutch design in which we induced females to produce more eggs (8–10 eggs) than they normally would (6 or 7 eggs) to determine whether maternal effort and the allocation of resources to supernumerary eggs (those laid beyond the usual number) influence the offspring sex ratio. At the clutch level, we predicted that high-quality females, as defined by their ability to produce supernumerary eggs in response to egg removal, would overproduce sons relative to females treated the same way but producing fewer eggs. At the level of the egg, we predicted that supernumerary eggs would more likely contain daughters than sons. As predicted, females producing extralarge clutches overproduced sons and those producing smaller clutches produced relatively more daughters. Last-laid eggs were also more likely to contain daughters than earlier-laid eggs although there was no difference in the mass of eggs containing males and females. These results suggest that mothers adjust the sex of their offspring strategically to maximize fitness.

Adult male and female birds typically respond differently to immunological challenges, but whether this difference is present in altricial nestlings is not well-documented. Furthermore, the timing of the development of different axes of the immune system might vary in nestlings and also be affected by differences in condition and health state. We tested for sex-related differences in the immune response of nestling House Wrens (Troglodytes aedon Vieillot, 1809) to the injection of phytohaemagglutinin (PHA) and in the bacteria-killing capacity of their plasma. Based on prior work, we predicted that if there were sex-related differences in immune responsiveness, they would most likely occur when the sexes differed in some measure of condition or health state. Female nestlings had a stronger response to the injection of PHA than males in the one of three breeding seasons in which the condition of nestling females exceeded that of males, suggesting that the response to PHA injection is condition-dependent rather than sex-dependent. The sexes, however, did not differ in bactericidal activity, suggesting that different axes of the immune system may not be affected to the same degree by differences in sex or in condition.

WhiB-like (Wbl) proteins are well known for their diverse roles in actinobacterial morphogenesis, cell division, virulence, primary and secondary metabolism, and intrinsic antibiotic resistance. Gene disruption experiments showed that three different Actinobacteria (Mycobacterium smegmatis, Streptomyces lividans, and Rhodococcus jostii) each exhibited a different whiB7-dependent resistance profile. Heterologous expression of whiB7 genes showed these resistance profiles reflected the host's repertoire of endogenous whiB7-dependent genes. Transcriptional activation of two resistance genes in the whiB7 regulon, tap (a multidrug transporter) and erm(37) (a ribosomal methyltransferase), required interaction of WhiB7 with their promoters. Furthermore, heterologous expression of tap genes isolated from Mycobacterium species demonstrated that divergencies in drug specificity of homologous structural proteins contribute to the variation of WhiB7-dependent drug resistance. WhiB7 has a specific tryptophan/glycine-rich region and four conserved cysteine residues; it also has a peptide sequence (AT-hook) at its C terminus that binds AT-rich DNA sequence motifs upstream of the promoters it activates. Targeted mutagenesis showed that these motifs were required to provide antibiotic resistance in vivo. Anaerobically purified WhiB7 from S. lividans was dimeric and contained 2.1 ± 0.3 and 2.2 ± 0.3 mol of iron and sulfur, respectively, per protomer (consistent with the presence of a 2Fe-2S cluster). However, the properties of the dimer's absorption spectrum were most consistent with the presence of an oxygen-labile 4Fe-4S cluster, suggesting 50% occupancy. These data provide the first insights into WhiB7 iron-sulfur clusters as they exist in vivo, a major unresolved issue in studies of Wbl proteins.

Tuberculosis therapeutic options are limited by the high intrinsic antibiotic resistance of Mycobacterium tuberculosis. The putative transcriptional regulator WhiB7 is crucial for the activation of systems that provide resistance to diverse antibiotic classes. Here, we used in vitro run-off, two-hybrid assays, as well as mutagenic, complementation and protein pull-down experiments, to characterize WhiB7 as an auto-regulatory, redox-sensitive transcriptional activator in Mycobacterium smegmatis. We provide the first direct biochemical proof that a WhiB protein promotes transcription and also demonstrate that this activity is sensitive to oxidation (diamide). Its partner protein for transcriptional activation was identified as SigA, the primary sigma factor subunit of RNA polymerase. Residues required for the interaction mapped to region 4 of SigA (including R515H) or adjacent domains of WhiB7 (including E63D). WhiB7’s ability to provide a specific spectrum of antibiotic-resistance was dependent on these residues as well as its C-terminal AT-hook module that binds to an AT-rich motif immediately upstream of the −35 hexamer recognized by SigA. These experimentally established constrains, combined with protein structure predictions, were used to generate a working model of the WhiB7–SigA-promoter complex. Inhibitors preventing WhiB7 interactions could allow the use of previously ineffective antibiotics for treatment of mycobacterial diseases.

While modern cephalosporins developed for broad spectrum antibacterial activities have never been pursued for tuberculosis (TB) therapy, we identified first generation cephalosporins having clinically relevant inhibitory concentrations, both alone and in synergistic drug combinations. Common chemical patterns required for activity against Mycobacterium tuberculosis were identified using structure-activity relationships (SAR) studies. Numerous cephalosporins were synergistic with rifampicin, the cornerstone drug for TB therapy and ethambutol, a first-line anti-TB drug. Synergy was observed even under intracellular growth conditions where beta-lactams typically have limited activities. Cephalosporins and rifampicin were 4- to 64-fold more active in combination than either drug alone; however, limited synergy was observed with rifapentine or rifabutin. Clavulanate was a key synergistic partner in triple combinations. Cephalosporins (and other beta-lactams) together with clavulanate rescued the activity of rifampicin against a rifampicin resistant strain. Synergy was not due exclusively to increased rifampicin accumulation within the mycobacterial cells. Cephalosporins were also synergistic with new anti-TB drugs such as bedaquiline and delamanid. Studies will be needed to validate their in vivo activities. However, the fact that cephalosporins are orally bioavailable with good safety profiles, together with their anti-mycobacterial activities reported here, suggest that they could be repurposed within new combinatorial TB therapies.

Glucocorticoids circulating in breeding birds during egg production accumulate within eggs, and may provide a potent form of maternal effect on offspring phenotype. However, whether these steroids affect offspring development remains unclear. Here, we employed a non-invasive technique that experimentally elevated the maternal transfer of corticosterone to eggs in a wild population of house wrens. Feeding corticosterone-injected mealworms to free-living females prior to and during egg production increased the number of eggs that females produced and increased corticosterone concentrations in egg yolks. This treatment also resulted in an increase in the amount of yolk allocated to eggs. Offspring hatching from these eggs begged for food at a higher rate than control offspring and eventually attained increased prefledging body condition, a trait predictive of their probability of recruitment as breeding adults in the study population. Our results indicate that an increase in maternal glucocorticoids within the physiological range can enhance maternal investment and offspring development.

Four butenolides, ramariolides A-D (1-4), have been isolated from the fruiting bodies of the coral mushroom Ramaria cystidiophora. Their structures were elucidated by analysis of 1D and 2D NMR data and a single-crystal X-ray diffraction analysis of 1, and their absolute configurations were established using Mosher's method. The major metabolite, ramariolide A (1), which contains an unusual spiro oxiranebutenolide moiety, showed in vitro antimicrobial activity against Mycobacterium smegmatis and Mycobacterium tuberculosis.

Current tuberculosis treatment regimens are notoriously limited, lengthy and becoming increasingly ineffective due to the emergence of drug-resistant mutant strains of Mycobacterium tuberculosis. The intrinsic resistance of M. tuberculosis to the majority of available drugs relies both on the impermeability of its cell envelope, and its ability to activate specific genes and physiological states. WhiB7 is a transcriptional regulatory protein underlying this adaptive process. Transcription of the whiB7 gene is upregulated in response to a variety of antibiotics having different structures and targets, as well as in response to metabolic signals. The whiB7 regulon activates various systems of intrinsic drug resistance involving antibiotic export, antibiotic inactivation (by chemical modifications of the drug or its target) and significant changes to thiol redox balance. Drugs have been identified that inactivate resistance determinants in the whiB7 regulon, thereby potentiating the activities of diverse antibiotics against M. tuberculosis.

Behavioural syndromes have been identified in a large number of species, yet our understanding of them in an ecological context remains poor. Specifically, there are few data that relate behavioural syndromes to other biologically important behaviours and, ultimately, to reproductive success. In this field study, we examined the aggressiveness and boldness of free-living male house wrens (Troglodytes aedon) and found a statistically significant positive relationship between these two behaviours (i.e. a behavioural syndrome). When we examined the two axes of this behavioural syndrome in relation to the male’s share of provisioning, we found a negative relation between a male’s aggressiveness and his provisioning rate, but no relationship between male boldness and provisioning behaviour. These differences in provisioning behaviour among males with different levels of aggression may reflect differences in reproductive strategies or different life-history trade-offs among males. Moreover, these results indicate that while some behavioural traits may be correlated, this does not mean that traits that compose the behavioural syndrome cannot evolve independently of one another.

The reproductive costs associated with the upregulation of immunity have been well-documented and constitute a fundamental trade-off between reproduction and self-maintenance. However, recent experimental work suggests that parents may increase their reproductive effort following immunostimulation as a form of terminal parental investment as prospects for future reproduction decline. We tested the trade-off and terminal investment hypotheses in a wild population of house wrens ( Troglodytes aedon ) by challenging the immune system of breeding females with lipopolysaccharide, a potent but non-lethal antigen. Immunized females showed no evidence of reproductive costs; instead, they produced offspring of higher phenotypic quality, but in a sex-specific manner. Relative to control offspring, sons of immunized females had increased body mass and their sisters exhibited higher cutaneous immune responsiveness to phytohaemagglutinin injection, constituting an adaptive strategy of sex-biased allocation by immune-challenged females to enhance the reproductive value of their offspring. Thus, our results are consistent with the terminal investment hypothesis, and suggest that maternal immunization can induce pronounced transgenerational effects on offspring phenotypes.

Theory predicts that investment in current offspring should negatively influence a parent’s ability to invest in future offspring. Despite extensive interest in documenting reproductive costs in birds, evidence for fitness-related costs of egg production to breeding females is scarce. In this study, we used an egg-removal experiment on the house wren, Troglodytes aedon , to test the hypothesis that producing eggs is reproductively costly. By removing eggs from the nest as they were laid, we induced females to produce more eggs than normal, although experimental and control females incubated clutches of similar size. Females producing extra eggs paid steep fitness costs for their increased effort; relative to controls, females with increased egg-laying demands were less likely to reproduce again within the same breeding season, and those that did took longer to do so, had a smaller clutch size and produced fewer fledglings from their subsequent broods. Although females in each treatment group were equally likely to return to breed the following summer, experimental females produced fewer eggs than control females in their next breeding season. This is the first demonstration that increased effort in egg production reduces the future reproductive output of passerine birds.

Gene clusters found in bacterial species classified as Streptomyces encode the majority of known antibiotics as well as many pharmaceutically active compounds. A site-specific recombination system similar to those that mediate plasmid conjugation was engineered to catalyze tandem amplification of one of these gene clusters in a heterologous Streptomyces species. Three genetic elements were known to be required for DNA amplification in S. kanamyceticus : the oriT -like recombination sites RsA and RsB, and ZouA, a site-specific relaxase similar to TraA proteins that catalyze plasmid transfer. We inserted RsA and RsB sequences into the S. coelicolor genome flanking a cluster of 22 genes ( act ) responsible for biosynthesis of the polyketide antibiotic actinorhodin. Recombination between RsA and RsB generated zouA -dependent DNA amplification resulting in 4–12 tandem copies of the act gene cluster averaging nine repeats per genome. This resulted in a 20-fold increase in actinorhodin production compared with the parental strain. To determine whether the recombination event required taxon-specific genetic effectors or generalized bacterial recombination ( recA ), it was also analyzed in the heterologous host Escherichia coli . zouA was expressed under the control of an inducible promoter in wild-type and recA mutant strains. A plasmid was constructed with recombination sites RsA and RsB bordering a drug resistance marker. Induction of zouA expression generated hybrid RsB/RsA sites, evidence of site-specific recombination that occurred independently of recA . ZouA-mediated DNA amplification promises to be a valuable tool for increasing the activities of commercially important biosynthetic, degradative, and photosynthetic pathways in a wide variety of organisms.

The manipulating androgens hypothesis (MAH) proposes that female birds increase the level of testosterone (T) in their eggs to promote greater offspring begging, and thereby elicit increased provisioning by their mates. We examined the effect of a positive in ovo manipulation of T on provisioning by house wren, Troglodytes aedon, parents, and concomitantly examined the begging response of nestlings. We also examined the mass of nestlings throughout their growth to assess the effect of T on their development, and three measures of nest performance: hatching success, nestling survival and the proportion of nestlings that fledged. Nestlings hatching from T-injected eggs begged more than nestlings hatching from control (oil-injected) eggs early in the nestling period, but not later in the nestling period. However, treatment had no effect on the levels of parental provisioning or nestling mass gain, nor any effect on hatching success or nestling survival. There was a significant increase in parental provisioning rate, but a decline in the size of prey taken to the nest over the course of the breeding season, which was likely the result of declining environmental quality. Our results support neither the MAH, nor the expectation that nestlings should grow at different rates in relation to in ovo titres of T.

Pyridinium-based oxime compounds have been utilized worldwide as antidotes following exposure to anticholinesterase agents. In the event of combined chemical and biological incident, it is of vital importance to know the ability of antidotes to provide additional protection against biological threats. This paper reports results of in vitro antimicrobial and antiprotozoal activities of a series of quaternary pyridinium oximes against a number of lower pathogenicity BSL-1 and 2 agents. In general, our compound panel had little to no antimicrobial action except for thiophene- and benzothiophene-substituted monoquaternary pyridinium compounds 21 and 24 that showed moderate antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus with IC(50) values ranging from 12.2 to 17.7 μg/mL. Compounds 21 and 24 also exhibited antileishmanial activity against Leishmania donovani with IC(50) values of 19 and 18 μg/mL, respectively. Another monoquaternary pyridinium compound with a bromobutyl side chain 17 showed antimalarial activity against both a chloroquine sensitive and resistant strains of Plasmodium falciparum with IC(50) values of 3.7 and 4.0 μg/mL, respectively. None of the bisquaternary pyridinium compounds showed antimicrobial or antiprotozoal activity. None of the compounds showed cytotoxic effects toward mammalian kidney fibroblasts. Results of this study indicate that the pyridinium compounds, some of which are already in use as antidotes, do not have significant antimicrobial and antiprotozoal activities and cannot be relied upon for additional protection in the event of combined chemical-biological incident.

Immunological measures are increasingly being applied to ecological and evolutionary studies of wild vertebrates, yet frequently it is not clear how condition and environmental factors correlate with various immune parameters. We used mixed-model ANOVA to examine the effects of several measures of condition (both morphological and physiological) and environmental factors on two measures of immune responsiveness in nestling house wrens (Troglodytes aedon L.) to test the hypothesis that nestlings in good condition mount stronger immune responses than those in poor condition. Based on previous studies, we predicted that the innate bactericidal response would be less likely to be affected by condition-related factors than the cutaneous response, which includes both innate and the more costly adaptive components. Both cutaneous immune activity (i.e., phytohaemagglutinin [PHA] response) and innate immune responsiveness (i.e., plasma bactericidal activity) varied significantly among broods. Nestling PHA response was significantly influenced by year, mass, and the time of day that the challenge was administered. However, besides nest of origin, no other variable examined had a significant effect on bactericidal activity. Morphological condition, assessed as body mass adjusted for structural size, differed significantly among nests and years and was positively correlated with hematocrit but not plasma albumin/gamma-globulin proteins, indicating that these are measures of different aspects of health state.

Female reproductive investment can vary according to their mate’s attractiveness, and males may differentially invest according to their own attractiveness. Thus, when studying female parental investment, male investment must also be considered. We tested the hypothesis that the attractiveness of male house wrens (Troglodytes aedon) influences their investment independent of their own intrinsic quality by manipulating the number of nest sites (one ¼ control; 4 ¼ attractive) in each male’s territory. Treatments (attractive or control) were applied prior to (natural state) or after (imposed state) male settlement, and male investment was determined twice during the nestling stage by the number of trips males made to the nest to provision their nestlings. Males that settled in the attractive territories were significantly older than those that settled in control territories in the natural state. There was a significant interaction effect between state and treatment on male provisioning. Provisioning rates of attractive and control males in the imposed state did not differ, but attractive males in the natural state provisioned at a lower rate than control males late in the nestling stage. Thus, provisioning by males is influenced more by their intrinsic quality than their attractiveness. Key words: attractiveness, house wren, mate choice, provisioning, reproductive allocation, Troglodytes aedon. [Behav Ecol]

The inhibition of recombinant mouse acetylcholinesterase (rMAChE) and electric eel acetylcholinesterase (EEAChE) by seven, structurally different chromophore-based (dansyl, pyrene, dabsyl, diethylamino- and methoxycoumarin, Lissamine rhodamine B, and Texas Red) propargyl carboxamides or sulfonamides was studied. Diethylaminocoumarin, Lissamine, and Texas Red amides inhibited rMAChE with IC50 values of 1.00 microM, 0.05 microM, and 0.70 microM, respectively. Lissamine and Texas Red amides inhibited EEAChE with IC50 values of 3.57 and 10.4 microM, respectively. The other chromophore amides did not inhibit either AChE. The surprising inhibitory potency of Lissamine was examined in further detail against EEAChE and revealed a mixed-type inhibition with Ki = 11.7 microM (competitive) and Ki' = 24.9 microM (noncompetitive), suggesting that Lissamine binds to free enzyme and enzyme-substrate complex.

The pathogenicity of mycobacterial infections depends on virulence factors that mediate survival inside host macrophages. These virulence factors are generally believed to be specific for pathogenic species and absent or mutated in non-pathogenic strains. The serine/threonine protein kinase G (PknG) mediates survival of mycobacteria within macrophages by blocking lysosomal delivery. Here we describe a gene of the non-pathogenic species Mycobacterium smegmatis that is 78% identical with pknG of Mycobacterium tuberculosis and M. bovis bacillus Calmette-Guerin (BCG). When cloned into expression vectors, the M. smegmatis pknG orthologue produced an active kinase and performed the same function as its M. bovis BCG counterpart in intracellular survival. In addition, similar levels of pknG transcripts were found in M. bovis BCG and M. smegmatis. However, virtually no translation product of chromosomal pknG could be detected in M. smegmatis both after in vitro growth and after macrophage infection. This lack of efficient translation was shown to be caused by regulatory elements in the upstream region of the M. smegmatis gene. The data reveal dramatically increased translational efficiency of a virulence gene in a pathogenic mycobacterium compared with a non-pathogenic mycobacterium suggesting that changes in expression levels may underlie evolution of pknG and other pathogenicity genes in mycobacterium.

Proteolysis, a regulated biological process, is reflected by protein spot molecular weight distribution in 2-D gel electrophoretograms. Here we report studies of Streptomyces cultures as they undergo two different developmental processes involving proteolysis. Systematic changes in protein molecular weight distribution between the control samples and those with high activity of proteases were demonstrated. The observations were supported by a numerical model of degradation and its influence on the M(r) distribution. Simple statistics could be used to distinguish between normal and degradative 2-D gel electrophoretic patterns.

Summary Among the unusual breeding habits of the non-parasitic Yellow-billed and Black-billed Cuckoos of North America are great variability in clutch size and rate of laying, initiation of incubation long before the clutch is complete, occasional laying in nests of other species, annual irregularity in the timing of the breeding season, and semi-nomadic post-migratory movements into breeding areas where food is abundant. These facts, in addition to their peculiar diet and the very large size of their eggs, suggest that cuckoos have extraordinary problems in obtaining adequate energy for reproduction. At Bloomington, Indiana (U.S.A.), during a 15-year period, anomalies in the reproductive activities of cuckoos were concentrated into two years in which food was abundant. This was particularly true of one of these years, when there was a vast emergence of periodical cicadas: the Yellow-billed Cuckoo advanced its normal schedule and bred during peak cicada abundance, laid unusually large clutches, and parasitized Black-billed Cuckoo nests. Some females may have resumed laying in nests in which, having already deposited clutches of normal size, they had been incubating for long periods; the alternative possibility is that there was intraspecific brood parasitism. The erratic egg-laying behaviour of these cuckoos is attributed to the evolution of mechanisms permitting very quick exploitation of a favourable feeding situation. It is suggested that reproductive behaviour has become so responsive to an abundance of food that normal ordering and integration of the stages of breeding have been lost in some females. Such a loss could be responsible for the laying of eggs in alien nests, and it may have been the antecedent of obligate brood parasitism in parasitic cuckoo species.

Attempts to characterize recombinant integral membrane proteins (IMPs) by mass spectrometry are frequently hindered by several factors including the detergents required for extraction and purification that interferes with analysis, poor solubility, incomplete digestion, and limited identification of the transmembrane domain-spanning peptides. The goal of this study was to examine and develop methods for purification of an IMP that are amenable to downstream digestion of the protein and peptide analysis by mass spectrometry. In this study, we have overexpressed a candidate IMP, the vesicular glutamate transporter 1 (VGLUT1) in Pichia pastoris and examined conditions for the efficient affinity purification, in-solution digestion, and analysis of the protein. Analysis of the intact purified protein without detergent was performed by MALDI-TOF mass spectrometry. The purified IMP was digested with trypsin, and the resulting peptides were identified. A method that utilizes differential solubility and ionization properties of hydrophobic and hydrophilic peptides was developed. Large hydrophobic peptides were only detected in solutions containing 50% formic acid. Ionization of hydrophilic peptides was suppressed in formic acid, but they produced a strong signal in 50% acetonitrile. Eighty-seven percent sequence coverage of the protein was obtained with only one large hydrophobic peptide that remained unidentified. The results demonstrate a simple method to purify and digest a recombinant IMP for analysis by mass spectrometry.

This paper describes a comparative systems level analysis of the developmental proteome and transcriptome in the model antibiotic-producing eubacterium Streptomyces coelicolor, cultured on different media. The analysis formulates expression as the superposition of effects of regulatory networks and biological processes which can be identified using singular value decomposition (SVD) of a data matrix formed by time series measurements of expression of individual genes throughout the cell cycle of the bacterium. SVD produces linearly orthogonal factors, each of which can represent an independent system behavior defined by a linear combination of the genes/proteins highly correlated with the corresponding factor. By using SVD of the developmental time series of gene expression, as measured by both protein and RNA levels, we show that on the highest level of control (representing the basic kinetic behavior of the population), the results are identical, regardless of the type of experiment or cultivation method. The results show that this approach is capable of identifying basic regulatory processes independent of the environment in which the organism lives. It also shows that these processes are manifested equally on protein and RNA levels. Biological interpretation of the correlation of the genes and proteins with significant eigenprofiles (representing the highest level kinetic behavior of protein and/or RNA synthesis) revealed their association with metabolic processes, stress responses, starvation, and secondary metabolite production.

A comprehensive analysis was done to evaluate the potential use of anti-parasitic macrocyclic lactones (including avermectins and milbemycins) for Buruli ulcer (BU) therapy. A panel containing nearly all macrocyclic lactones used in human or in veterinary medicine was analyzed for activity in vitro against clinical isolates of Mycobacterium ulcerans. Milbemycin oxime and selamectin were the most active drugs against M. ulcerans with MIC values from 2 to 8 μg/mL and 2 to 4 μg/mL, respectively. In contrast, ivermectin and moxidectin, which are both in clinical use, showed no significant activity (MIC> 32 μg/mL). Time-kill kinetic assays showed bactericidal activity of selamectin and in vitro pharmacodynamic studies demonstrated exposure-dependent activity. These data together with analyses of published pharmacokinetic information strongly suggest that selamectin is the most promising macrocyclic lactone for BU treatment., Author Summary Buruli ulcer (BU) is a chronic debilitating mycobacterial disease of the skin and soft tissue caused by Mycobacterium ulcerans. It is mainly found in tropical regions and often linked to poverty. BU can be cured in most cases with the standard treatment, a combination of rifampicin and the injectable antibiotic streptomycin. However, new optimized treatment regimens are needed, especially to prepare for an eventual development of resistance to rifampicin, the most efficacious drug for BU therapy. Since traditional antibacterial drug discovery is not a practical option for BU, using approved drugs for alternative clinical indications would be a more economical and faster way to implement new anti-BU therapies. We reported previously that anti-parasitic avermectins are active against Mycobacterium tuberculosis. Here we show that some are also active in vitro against other mycobacterial species, including M. marinum and M. ulcerans. In this study, we undertook a comprehensive approach to evaluate additional macrocyclic lactones including compounds used in veterinary medicine. Based on our in vitro measurements of their activities and a literature review of their pharmacokinetic properties, we present strong arguments that selamectin is the avermectin with the highest potential for being repurposed for BU treatment.

Trade-offs in the allocation of finite resources among different stages of a breeding attempt as well as between different reproductive events should shape the evolution of life-history traits. To investigate the effects of incubation effort on within-brood and between-brood trade-offs in house wrens (Troglodytes aedon), we manipulated the clutch size that females incubated. We isolated effects of incubation by reversing the manipulation at hatching to allow all parents to provision their natural brood sizes. Females that incubated enlarged clutches had longer incubation periods than control females, both early and late in the season, suggesting that the experimental treatment increased incubation effort. Contrary to predictions, however, increased incubation effort did not adversely affect the allocation of effort to nestling provisioning. Rather, in the early season, but not in the late season, females that incubated enlarged clutches appeared to allocate more effort to nestling provisioning, producing heavier and larger fledglings than control females. Although females with enlarged early-season clutches consequently lost more mass than control females, this was likely an adaptive response to reduce wing loading in anticipation of high provisioning demands. There were no treatment-related differences in fledgling mass or size, or in female mass loss, in the late season. Thus, elevated incubation demands negatively affected a fitness-related trait (duration of incubation) that may constrain clutch size but not the allocation of resources to subsequent stages of the same breeding event or to subsequent breeding events. We suggest that environmental conditions may mediate clutch-size effects on trade-offs in allocation of resources between incubation and nestling provisioning. Key words: clutch size, house wren, incubation, life history, trade-offs, Troglodytes aedon. [Behav Ecol]

The intrinsic resistance of Mycobacterium tuberculosis and related pathogens to most common antibiotics limits chemotherapeutic options to treat tuberculosis and other mycobacterial diseases. Resistance has traditionally been attributed to the unusual multi-layer cell envelope that functions as an effective barrier to the penetration of antibiotics. Recent insights into mechanisms that neutralize the toxicity of antibiotics in the cytoplasm have revealed systems that function in synergy with the permeability barrier to provide intrinsic resistance. Here, we highlight the growing pool of information about internal, antibiotic-responsive regulatory proteins and corresponding resistance genes, and present new concepts that rationalize how they might have evolved. Pharmaceutical inhibition of these intrinsic systems could make many previously available antibiotics active against M. tuberculosis.

When male birds defend all-purpose breeding territories, females may select mates based on indicators of male or territory quality, or both. However, in non-experimental studies, it can be difficult to determine which traits females prefer because measures of male and territory quality frequently covary. We conducted a series of studies to investigate the traits female house wrens (Troglodytes aedon) use to select a social mate when each male is provided with nest sites of equal quality (i.e., nestboxes). We first compared the phenotypic and territory vegetation traits of males with the time it took them to secure a social mate after returning from the wintering grounds. Male arrival and pairing date were positively correlated and early-arriving males were in better condition and occupied territories in more preferred vegetation than late-arriving males. To test the hypothesis that early-arriving males possessed phenotypic or territory traits that led to their pairing more quickly than late-arriving males, we removed all females after pairs had been established and determined male success in attracting new mates. Male settlement and pairing date with replacement females were not correlated as had been male settlement and pairing date with original females, and male time-to-pairing was not correlated with male song rate, condition index, or territory vegetation. When we manipulated the abundance of nest sites on territories, after males settled but before females arrived, male time-to-pairing, independent of male condition index or territory vegetation, was negatively related to the number of nest sites on a male's territory. These results suggest that under natural conditions females rely on nest-site quantity and, likely, quality when selecting a social mate.

Chemotherapeutic options to treat tuberculosis are severely restricted by the intrinsic resistance of Mycobacterium tuberculosis to the majority of clinically applied antibiotics. Such resistance is partially provided by the low permeability of their unique cell envelope. Here we describe a complementary system that coordinates resistance to drugs that have penetrated the envelope, allowing mycobacteria to tolerate diverse classes of antibiotics that inhibit cytoplasmic targets. This system depends on whiB7 , a gene that pathogenic Mycobacterium shares with Streptomyces , a phylogenetically related genus known as the source of diverse antibiotics. In M. tuberculosis , whiB7 is induced by subinhibitory concentrations of antibiotics (erythromycin, tetracycline, and streptomycin) and whiB7 null mutants ( Streptomyces and Mycobacterium ) are hypersusceptible to antibiotics in vitro . M. tuberculosis is also antibiotic sensitive within a monocyte model system. In addition to antibiotics, whiB7 is induced by exposure to fatty acids that pathogenic Mycobacterium species may accumulate internally or encounter within eukaryotic hosts during infection. Gene expression profiling analyses demonstrate that whiB7 transcription determines drug resistance by activating expression of a regulon including genes involved in ribosomal protection and antibiotic efflux. Components of the whiB7 system may serve as attractive targets for the identification of inhibitors that render M. tuberculosis or multidrug-resistant derivatives more antibiotic-sensitive.

Males in the cavity-nesting house wren (Troglodytes aedon) frequently add arthropod cocoons to their nests during building, possibly as an ornamental cue for female choice. We tested this hypothesis by comparing the time to pairing for males that did and did not add cocoons to their nests and for males in whose nests we manipulated the number of cocoons prior to pairing. We also tested the hypothesis that females acquire fitness-related benefits by selecting mates based on their use of cocoons. The use of cocoons by males was not consistently related to habitat, but the number of cocoons added per nest increased during the course of the breeding season. Contrary to prediction, the time to pairing for males adding cocoons was significantly longer than that for males without cocoons in their nests at both unmanipulated and experimental nests. There was also no consistent fitnessrelated benefit for females related to the use of cocoons by their mates. Therefore, we conclude that females did not prefer males that added cocoons to their nests, and that the increased time to pairing for males that add cocoons likely results in fitnessrelated costs brought about by delayed breeding. Nonetheless, male house wrens routinely use cocoons, and why they do so remains unknown. Key words: female choice, house wrens, nest building, sexual selection, Troglodytes aedon. [Behav Ecol]

Acoustic measurements at 1.5–5 kHz on fish in the Gulf of Maine showed a swimbladder-resonance peak near 2.5 kHz at 160–190-m depth. Midwater trawls confirmed that the fish were likely to be Atlantic herring (Clupea harengus) of 19–29 cm length. Calculation using a model of swimbladder resonance gives swimbladder volumes of 1.2% of fish weight at 160–190 m. Extrapolation of this volume of gas using Boyle's Law suggests that at the sea surface, these herring would need to inflate their swimbladders by up to five to six times the volume required for neutral buoyancy. If these fish were to maintain this volume of gas with surface “gulping”, they would need to submerge from the sea surface with a 30% excess buoyancy. In general, swimbladders of the Clupeidae may have greater volumes of gas than if the fish were neutrally buoyant at the sea surface and the interpretation of HF-echosounder surveys may be additionally complex when the volume of gas and swimbladder volume are difficult to predict. Mechanisms of how herring obtain additional swimbladder gas are discussed.

We identified adpA as an araC -like regulatory gene needed for colonial morphogenesis in Streptomyces coelicolor and showed that its activity depended on a unique TTA triplet corresponding to the leucyl-tRNA gene ( bldA ). These findings partially explained the dependence of aerial mycelium formation on a rare tRNA that is postulated to have developmental control functions.

Bacteria typically undergo intermittent periods of starvation and adaptation, emulated as diauxic growth in the laboratory. In association with growth arrest elicited by metabolic stress, the differentiating eubacterium Streptomyces coelicolor not only adapts its primary metabolism, but can also activate developmental programmes leading to morphogenesis and antibiotic biosynthesis. Here, we report combined proteomic and metabolomic data of S. coelicolor used to analyse global changes in gene expression during diauxic growth in a defined liquid medium. Cultures initially grew on glutamate, providing the nitrogen source and feeding carbon (as 2-oxoglutarate) into the TCA cycle, followed by a diauxic delay allowing reorientation of metabolism and a second round of growth supported by NH4+, formed during prediauxic phase, and maltose, a glycolytic substrate. Cultures finally entered stationary phase as a result of nitrogen starvation. These four physiological states had previously been defined statistically by their distinct patterns of protein synthesis and heat shock responses. Together, these data demonstrated that the rates of synthesis of heat shock proteins are determined not only by temperature increase but also by the patterns and rates of metabolic flux in certain pathways. Synthesis profiles for metabolic- and stress-induced proteins can now be interpreted by the identification of 204 spots (SWICZ database presented at http://proteom.biomed.cas.cz). Cluster analysis showed that the activity of central metabolic enzymes involved in glycolysis, the TCA cycle, starvation or proteolysis each displayed identifiable patterns of synthesis that logically underlie the metabolic state of the culture. Diauxic lag was accompanied by a structured regulatory programme involving the sequential activation of heat-, salt-, cold- and bacteriostatic antibiotic (pristinamycin I, PI)-induced stimulons. Although stress stimulons presumably provide protection during environmental- or starvation-induced stress, their identities did not reveal any coherent adaptive or developmental functions. These studies revealed interactive regulation of metabolic and stress response systems including some proteins known to support developmental programmes in S. coelicolor.

The TipAL protein, a bacterial transcriptional regulator of the MerR family, is activated by numerous cyclic thiopeptide antibiotics. Its C-terminal drug-binding domain, TipAS, defines a subfamily of broadly distributed bacterial proteins including Mta, a central regulator of multidrug resistance in Bacillus subtilis. The structure of apo TipAS, solved by solution NMR [Brookhaven Protein Data Bank entry 1NY9], is composed of a globin-like alpha-helical fold with a deep surface cleft and an unfolded N-terminal region. Antibiotics bind within the cleft at a position that is close to the corresponding heme pocket in myo- and hemoglobin, and induce folding of the N-terminus. Thus the classical globin fold is well adapted not only for accommodating its canonical cofactors, heme and other tetrapyrroles, but also for the recognition of a variety of antibiotics where ligand binding leads to transcriptional activation and drug resistance.

Summary Whereas in Bacillus subtilis , a general stress response stimulon under the control of a single sigma factor (SigB) is induced by different physiological and environmental stresses (heat, salt or ethanol shock), in Streptomyces coelicolor , these environmental stresses induce independent sets of proteins, and its genome encodes nine SigB paralogues. To investigate possible functions of multiple sigB -like genes in S. coelicolor , Pctc , a promoter routinely used to assay SigB activity in vivo , was analysed as a heterologous reporter. The fact that Pctc was activated by osmotic shock, but not by heat or ethanol, confirmed that stress response system(s) could operate indepen- dently in S. coelicolor . Pctc was also induced tran- siently during growth of liquid cultures, presumably by nutritional signals. We purified an RNA polymerase holoenzyme from crude extracts that catalysed spe- cific transcription of Pctc in vitro . Its sigma subunit was identified as a product of the sigH gene, which is co-transcribed downstream of a putative antisigma factor gene ( prsH ). Although the sigH function was not needed for normal colony morphology, prsH was conditionally required for both aerial hyphae forma- tion and regulation of antibiotic biosynthesis. Levels of two different sigH -encoded proteins were growth phase dependent but not significantly changed by osmotic stress, implying the important roles of post- translational regulatory elements such as PrsH. In addition, synthesis of three other SigH-like proteins was induced by osmotic stress, but not by ethanol or heat. Two of them were genetically assigned to sigH homologous loci sigI and sigJ and shown to be inde- pendently regulated. This family of SigH-like proteins displayed different osmotic response kinetics. Thus, in contrast to many other bacteria, S. coelicolor uses an osmotic sensory system that can co-ordinate the activity of multiple paralogues to control the relative activity of promoters within the same stress stimulon. Such specialized stress response systems may reflect adaptive functions needed for colonial differentiation.

In the multicellular bacterium Streptomyces coelicolor, functions of developmental (bald) genes are required for the biosynthesis of SapB, a hydrophobic peptidic morphogen that facilitates aerial hyphae formation. Here, we show that aerial hyphal growth and SapB biosynthesis could be activated independently from the normal developmental cascade by providing unprogrammed expression of functionally interactive genes within the ram cluster. ramC, ramS and ramR were essential for normal growth of aerial hyphae, and ramR, a response regulator gene, was a key activator of development. The ramR gene restored growth of aerial hyphae and SapB formation in all bald strains tested (albeit only weakly in the bldC mutant), many of which are characterized by physiological defects. Disruption of the ramR gene abolished SapB biosynthesis and severely delayed growth of aerial hyphae. Transcription of ramR was developmentally controlled, and RamR function in vivo depended on its putative phosphorylation site (D53). We identified and mapped RamR targets immediately upstream of the region encoding ramC and ramS, a putative operon. Overexpression of ramR in the wild-type strain increased SapB levels and caused a distinctive wrinkled surface topology. Based on these results, we propose that phenotypes of bald mutations reflect an early stage in the Streptomyces developmental programme similar to the spo0 mutations in the unicellular bacterium Bacillus subtilis, and that RamR has analogies to Spo0A, the Bacillus response regulator that integrates physiological signals before triggering endospore formation.

TipA is a transcriptional regulator found in diverse bacteria. It constitutes a minimal autoregulated multidrug resistance system against numerous thiopeptide antibiotics. Here we report the structures of its drug-binding domain TipAS in complexes with promothiocin A and nosiheptide, and a model of the thiostrepton complex. Drug binding induces a large transition from a partially unfolded to a globin-like structure. The structures rationalize the mechanism of promiscuous, yet specific, drug recognition: (i) a four-ring motif present in all known TipA-inducing antibiotics is recognized specifically by conserved TipAS amino acids; and (ii) the variable part of the antibiotic is accommodated within a flexible cleft that rigidifies upon drug binding. Remarkably, the identified four-ring motif is also the major interacting part of the antibiotic with the ribosome. Hence the TipA multidrug resistance mechanism is directed against the same chemical motif that inhibits protein synthesis. The observed identity of chemical motifs responsible for antibiotic function and resistance may be a general principle and could help to better define new leads for antibiotics.

In Streptomyces, a family of related butyrolactones and their corresponding receptor proteins serve as quorum-sensing systems that can activate morphological development and antibiotic biosynthesis. Streptomyces pristinaespiralis contains a gene cluster encoding enzymes and regulatory proteins for the biosynthesis of pristinamycin, a clinically important streptogramin antibiotic complex. One of these proteins, PapR1, belongs to a well known family of Streptomyces antibiotic regulatory proteins. Gel shift assays using crude cytoplasmic extracts detected SpbR, a developmentally regulated protein that bound to the papR1 promoter. SpbR was purified, and its gene was cloned using reverse genetics. spbR encoded a 25-kDa protein similar to Streptomyces autoregulatory proteins of the butyrolactone receptor family, including scbR from Streptomyces coelicolor. In Escherichia coli, purified SpbR and ScbR produced bound sequences immediately upstream of papR1, spbR, and scbR. SpbR DNA-binding activity was inhibited by an extracellular metabolite with chromatographic properties similar to those of the well known gamma-butyrolactone signaling compounds. DNase I protection assays mapped the SpbR-binding site in the papR1 promoter to a sequence homologous to other known butyrolactone autoregulatory elements. A nucleotide data base search showed that these binding motifs were primarily located upstream of genes encoding Streptomyces antibiotic regulatory proteins and butyrolactone receptors in various Streptomyces species. Disruption of the spbR gene in S. pristinaespiralis resulted in severe defects in growth, morphological differentiation, pristinamycin biosynthesis, and expression of a secreted superoxide dismutase.