Regulatory overlap and functional redundancy among Bacillus subtilis extracytoplasmic function sigma factors

Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma factors that regulate partially overlapping regulons related to cell envelope homeostasis and antibiotic resistance. Here, we investigated their physiological role by constructing a mutant set of single, double, triple, and quadruple ECF sigma factor deletions in the undomesticated B. subtilis strain NCIB3610. This mutant set was subsequently screened for defects in motility, multicellular differentiation, and sensitivity to more than 200 chemicals by using Phenotype MicroArrays. A quadruple mutant strain, harboring deletions of the sigV, sigY, sigZ, and ylaC gene, behaved indistinguishably from the wild-type strain, indicative of either regulatory redundancy or very specific functions of these four ECF sigma factors. In contrast, a triple mutant, inactivated for the sigM, sigW, and sigX genes (but none of the corresponding double mutants), showed a biphasic growth behavior and a complete loss of multicellular differentiation, as judged by both colony formation and the inability to form a pellicle. This triple mutant also displayed a greatly increased sensitivity to detergents and several cell wall antibiotics including beta-lactams, polymyxin B, and d-cycloserine. In several cases, these antibiotic-sensitive phenotypes are significantly enhanced in the triple mutant strain relative to strains lacking only one or two sigma factors.     

Robust assessment of associations between weather and eastern wild turkey nest success

Temperature and precipitation have been identified as factors that potentially influence eastern wild turkey (Meleagris gallopavo silvestris) reproduction, but robust analyses testing the relationship between weather parameters and turkey nest success are lacking. Therefore, we assessed how weather influenced turkey daily nest survival using 8 years of data collected from 715 nests across the southeastern United States. We also conducted exploratory analyses investigating if weather conditions during or prior to nesting best predicted nest success. We then assessed the possible implications of climate change through 2041–2060 for future eastern wild turkey daily nest survival and nest success for variables determined significant in analyses. During incubation, positive anomalies of minimum daily temperature were associated with greater daily nest survival. Precipitation during nesting was not a good predictor of daily nest survival. Exploratory analyses unexpectedly indicated that weather conditions in January prior to incubation were more important to nest success than weather conditions during incubation. In January, negative anomalies of minimum temperature and greater average daily precipitation were associated with greater nest success. Projections of future nest success or daily nest survival based on these relationships with the predictive covariates, and informed by climate models, suggest that nest success may increase as January precipitation increases and that daily nest survival may increase as temperature during incubation increases. These positive associations could be offset by a negative association between nest success and the expected increases in January minimum average temperature. Additional research is needed to investigate causes of these relationships and assess the implications of climate change for eastern wild turkey poult survival.     

Survival and cause-specific mortality of male wild turkeys across the southeastern United States

Estimating survival and cause-specific mortality of male eastern wild turkeys (Meleagris gallopavo silvestris) is important for understanding population dynamics and implementing appropriate harvest management. To better understand age-specific estimates of annual survival and harvest rates, we captured and marked male wild turkeys with leg bands (n = 311) or bands and transmitters (n = 549) in Georgia, Louisiana, North Carolina, and South Carolina, USA, during 2014–2022. We fitted time to event models to data from radio-marked birds to estimate cause-specific mortality and annual survival. We used band recovery models incorporating both band recovery and telemetry data to further investigate harvest rates and survival. Annual survival from known-fate models in hunted populations was 0.54 (95% CI = 0.49–0.59) for adults and 0.86 (95% CI = 0.81–0.92) for juveniles. Cause-specific mortality analysis produced an annual harvest estimate of 0.29 (95% CI = 0.24–0.33) for adults and 0.02 (95% CI = 0.01–0.03) for juveniles, whereas predation was 0.15 (95% CI = 0.10–0.20) and 0.12 (95% CI = 0.08–0.17), respectively. Annual survival for adult males in a non-hunted population was 0.83 (95% CI = 0.72–0.97). Survival rate was negatively correlated with harvest rate, indicating harvest was an additive mortality source. Annual survival from band recovery models was 0.40 (95% CI = 0.37–0.44) for adults and 0.88 (95% CI = 0.81– 0.93) for juveniles, whereas annual harvest estimates were 0.24 (95% CI = 0.23–0.25) for adults and 0.04 (95% CI = 0.03–0.05) for juveniles. Both models suggested no differences in annual survival across years or among study areas, which included privately owned and public properties. Harvest was an additive mortality source for male wild turkeys, suggesting that managers interested in increasing annual survival of adult males could consider ways of reducing harvest rates.     

In vitro mutagenesis of Bacillus subtilis by using a modified Tn7 transposon with an outward-facing inducible promoter

A Tn7 donor plasmid, pTn7SX, was constructed for use with the model gram-positive bacterium Bacillus subtilis. This new mini-Tn7, mTn7SX, contains a spectinomycin resistance cassette and an outward-facing, xylose-inducible promoter, thereby allowing for the regulated expression of genes downstream of the transposon. We demonstrate that mTn7SX inserts are obtained at a high frequency and occur randomly throughout the B. subtilis genome. The utility of this system was demonstrated by the selection of mutants with increased resistance to the antibiotic fosfomycin or duramycin.     

Modulation of surface antigens of a human monocyte cell line, U937, during incubation with T lymphocyte-conditioned medium: detection of T4 antigen and its presence on normal blood monocytes

The human monocyte line, U937, derived from an individual with histiocytic lymphoma, undergoes morphological and functional changes when incubated with medium conditioned by lectin-stimulated cloned human T lymphocytes. Using monoclonal antibodies and flow cytometry, we therefore analyzed alterations in surface components that might accompany these morphological changes, in comparison with components present on normal blood monocytes. The U937 cells possess three surface antigens in common with blood monocytes, detected with OKM1, 4F2, and anti-monocyte.2 (the last monocyte specific). DR antigen was not detectable on U937 cells with three anti-DR framework antibodies but was detected on blood monocytes. Unexpectedly, OKT4, a monoclonal antibody to T4 antigen previously believed to be restricted to helper T lymphocytes, also reacted with U937 cells. Six monoclonal antibodies to other epitopes on T4 also reacted with U937 cells. None of these could be inhibited by blocking of Fc receptors. T4 with its various epitopes were also expressed on normal human blood monocytes. Other lymphocyte surface markers (T3, T8, T6) and fibronectin were not detectable on U937 cells or monocytes. An individual, whose lymphocytes lacked the epitope detected with OKT4 but had epitopes detected with OKT4 A, B, C, and D, had monocytes with identical reactivity, evidence that the T4 on monocytes and lymphocytes are products of the same structural gene. Stimulation of U937 cells for 24 hours with supernatants from Con A-stimulated T lymphocyte clones caused an increase in expression of OKM1 and Fc receptor activity and a decrease in expression of T4, consistent with a more mature phenotype of blood monocytes. Although the function of the T4 molecule is unknown, it is notable that it is displayed by two cells of distinct lineage which interact in the response to soluble antigens.     

Neural correlates of threat perception: neural equivalence of conspecific and heterospecific mobbing calls is learned

Songbird auditory areas (i.e., CMM and NCM) are preferentially activated to playback of conspecific vocalizations relative to heterospecific and arbitrary noise. Here, we asked if the neural response to auditory stimulation is not simply preferential for conspecific vocalizations but also for the information conveyed by the vocalization. Black-capped chickadees use their chick-a-dee mobbing call to recruit conspecifics and other avian species to mob perched predators. Mobbing calls produced in response to smaller, higher-threat predators contain more "D" notes compared to those produced in response to larger, lower-threat predators and thus convey the degree of threat of predators. We specifically asked whether the neural response varies with the degree of threat conveyed by the mobbing calls of chickadees and whether the neural response is the same for actual predator calls that correspond to the degree of threat of the chickadee mobbing calls. Our results demonstrate that, as degree of threat increases in conspecific chickadee mobbing calls, there is a corresponding increase in immediate early gene (IEG) expression in telencephalic auditory areas. We also demonstrate that as the degree of threat increases for the heterospecific predator, there is a corresponding increase in IEG expression in the auditory areas. Furthermore, there was no significant difference in the amount IEG expression between conspecific mobbing calls or heterospecific predator calls that were the same degree of threat. In a second experiment, using hand-reared chickadees without predator experience, we found more IEG expression in response to mobbing calls than corresponding predator calls, indicating that degree of threat is learned. Our results demonstrate that degree of threat corresponds to neural activity in the auditory areas and that threat can be conveyed by different species signals and that these signals must be learned.     

Position-dependent effects on stability in tricyclo-DNA modified oligonucleotide duplexes

A series of oligodeoxyribonucleotides and oligoribonucleotides containing single and multiple tricyclo(tc)-nucleosides in various arrangements were prepared and the thermal and thermodynamic transition profiles of duplexes with complementary DNA and RNA evaluated. Tc-residues aligned in a non-continuous fashion in an RNA strand significantly decrease affinity to complementary RNA and DNA, mostly as a consequence of a loss of pairing enthalpy ΔH. Arranging the tc-residues in a continuous fashion rescues T_m and leads to higher DNA and RNA affinity. Substitution of oligodeoxyribonucleotides in the same way causes much less differences in T_m when paired to complementary DNA and leads to substantial increases in T_m when paired to complementary RNA. CD-spectroscopic investigations in combination with molecular dynamics simulations of duplexes with single modifications show that tc-residues in the RNA backbone distinctly influence the conformation of the neighboring nucleotides forcing them into higher energy conformations, while tc-residues in the DNA backbone seem to have negligible influence on the nearest neighbor conformations. These results rationalize the observed affinity differences and are of relevance for the design of tc-DNA containing oligonucleotides for applications in antisense or RNAi therapy.