Crystal structure of a bicupin protein HutD involved in histidine utilization in Pseudomonas.

Cupins form one of the most functionally diverse superfamilies of proteins, with members performing a wide range of catalytic, non-catalytic, and regulatory functions. HutD is a predicted bicupin protein that is involved in histidine utilization (Hut) in Pseudomonas species. Previous genetic analyses have suggested that it limits the upper level of Hut pathway expression, but its mechanism of action is unknown. Here, we have determined the structure of PfluHutD at 1.74 Å resolution in several crystallization conditions, and identified N-formyl-l-glutamate (FG, a Hut pathway intermediate) as a potential ligand in vivo. Proteins 2017; 85:1580-1588. © 2017 Wiley Periodicals, Inc.

The turnover of strains in intermittent and persistent nasal carriers of Staphylococcus aureus.

OBJECTIVES: We aimed to examine the dynamics of Staphylococcus aureus nasal carriage in healthy adults. METHOD: Selected S. aureus strains isolated from weekly nasal swabs obtained from 122 healthy young adults over a 13 week period were spa typed. RESULTS: The median duration of intermittent carriage was 4 weeks (IQR 2-6) and the median interval between episodes of carriage of different spa types was 3.5 weeks (IQR 2.25-4). 6/19 (32%) Persistent carriers were colonised with more than one spa type during the study, and in two persistent carriers a brief period of mixed colonisation with two spa types was observed. Even when the carriage strain changed, it was very rare for persistent carriers to have a period during which they were culture-negative (only 6/188 (3%) swabs submitted by persistent carriers failed to culture S. aureus). CONCLUSIONS: Our results imply that at least every eight weeks a healthy young adult is exposed to S. aureus sufficient to cause a new episode of carriage among intermittent carriers. Persistent carriers are almost always colonised with S. aureus and over the course of a year there will be at least one replacement of the dominant strain.

Testing temperature-induced proteomic changes in the plant-associated bacterium Pseudomonas fluorescens SBW25.

Traits used by bacteria to enhance ecological performance in natural environments are not well understood. Recognizing that the saprophytic plant-colonizing bacterium Pseudomonas fluorescens SBW25 experiences temperatures in its natural environment significantly cooler than the 28°C routinely used in the laboratory, we identified proteins differentially expressed between 28°C and the more environmentally relevant temperature of 14°C. Of 2102 protein isoforms, 32 were temperature responsive and identified by mass spectrometry. Seven of these (OmpR, MucD, GuaD, OsmY and three of unknown function, Tee1, Tee2 and Tee3) were selected for genetic and ecological analyses. In each instance, changes in protein expression with temperature were mirrored by parallel transcriptional changes. The fitness contribution of the genes encoding each of the seven proteins was larger at 14°C than 28°C and included two cases of trade-offs (enhanced fitness at one temperature and reduced fitness at the other -mucD and tee2 deletions). The relationship between the fitness effects of genes in vitro and in vivo was variable, but two temperature-responsive genes -osmY and mucD- contribute substantially to the ability of P. fluorescens to colonize the plant environment.

The structure-function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain.

The GGDEF response regulator WspR couples the chemosensory Wsp pathway to the overproduction of acetylated cellulose and cell attachment in the Pseudomonas fluorescens SBW25 wrinkly spreader (WS) genotype. Here, it is shown that WspR is a diguanylate cyclase (DGC), and that DGC activity is elevated in the WS genotype compared to that in the ancestral smooth (SM) genotype. A structure-function analysis of 120 wspR mutant alleles was employed to gain insight into the regulation and activity of WspR. Firstly, 44 random and defined pentapeptide insertions were produced in WspR, and the effects determined using assays based on colony morphology, attachment to surfaces and cellulose production. The effects of mutations within WspR were interpreted using a homology model, based on the crystal structure of Caulobacter crescentus PleD. Mutational analyses indicated that WspR activation occurs as a result of disruption of the interdomain interface, leading to the release of effector-domain repression by the N-terminal receiver domain. Quantification of attachment and cellulose production raised significant questions concerning the mechanisms of WspR function. The conserved RYGGEEF motif of WspR was also subjected to mutational analysis, and 76 single amino acid residue substitutions were tested for their effects on WspR function. The RYGGEEF motif of WspR is functionally conserved, with almost every mutation abolishing function.

Experimental adaptation to high and low quality environments under different scales of temporal variation.

We investigated the role of the scale of temporal variation in the evolution of generalism in populations of the bacterium Pseudomonas fluorescens. Replicate populations were propagated as batch cultures for approximately 1400 generations (192 days), in either high quality media only, low quality media only, or were alternated between the two at a range of temporal scales (between 1 and 48 days). Populations evolved in alternating media showed fitness increases in both media and the rate of alternation during selection had no effect on average fitness in either media. Moreover, the fitness of these populations in high quality media was the same as for populations evolved only in high quality media and likewise for low quality media. Populations evolved only in high or low quality media did not show fitness improvements in their nonselective media. These results indicate that cost-free generalists can evolve under a wide range of temporal variation.

Spatial heterogeneity and the stability of host-parasite coexistence.

Spatially heterogeneous environments can theoretically promote more stable coexistence of hosts and parasites by reducing the risk of parasite attack either through providing permanent spatial refuges or through providing ephemeral refuges by reducing dispersal. In experimental populations of Pseudomonas aeruginosa and the bacteriophage PP7, spatial heterogeneity promoted stable coexistence of host and parasite, while coexistence was significantly less stable in the homogeneous environment. Phage populations were found to be persisting on subpopulations of sensitive bacteria. Transferring populations to fresh microcosms every 24 h prevented the development of permanent spatial refuges. However, the lower dispersal rates in the heterogeneous environment were found to reduce parasite transmission thereby creating ephemeral refuges from phage attack. These results suggest that spatial heterogeneity can stabilize an otherwise unstable host-parasite interaction even in the absence of permanent spatial refuges.

Believe it or not--silver still poisons!

For centuries, silver has been endowed with therapeutic benefits. It is still used today as a "caustic" for superficial bleeding. Within 7days, we had 3 cases of "argyria" and then 2 more over the next month. The first 2 cases involved a husband and wife with a 3-y exposure to naturopathic hydrolyzed silver treatment. The third casewas a 37-y-old male in a state psychiatric facility noted to have darkly "discolored" skin probable obtained from herbal tea. The last 2 cases were a married couple into herbal medications who developed bluish discoloration of face and hands. Current cases due to "alternative medicine" may get worse as rumor reveals its popularity as prophylaxis against anthrax. The skin's grayish discoloration, made worse by sunlight, may persist for life.

Type III secretion in plant growth-promoting Pseudomonas fluorescens SBW25.

In vivo expression technology (IVET) analysis of rhizosphere-induced genes in the plant growth-promoting rhizobacterium (PGPR) Pseudomonas fluorescens SBW25 identified a homologue of the type III secretion system (TTSS) gene hrcC. The hrcC homologue resides within a 20-kb gene cluster that resembles the type III (Hrp) gene cluster of Pseudomonas syringae. The type III (Rsp) gene cluster in P. fluorescens SBW25 is flanked by a homologue of the P. syringae TTSS-secreted protein AvrE. P. fluorescens SBW25 is non-pathogenic and does not elicit the hypersensitive response (HR) in any host plant tested. However, strains constitutively expressing the rsp-specific sigma factor RspL elicit an AvrB-dependent HR in Arabidopsis thaliana ecotype Col-0, and a host-specific HR in Nicotiana clevelandii. The inability of wild-type P. fluorescens SBW25 to elicit a visible HR is therefore partly attributable to low expression of rsp genes in the leaf apoplast. DNA hybridization analysis indicates that rsp genes are present in many plant-colonizing Pseudomonas and PGPR, suggesting that TTSSs may have a significant role in the biology of PGPR. However, rsp and rsc mutants retain the ability to reach high population levels in the rhizosphere. While functionality of the TTSS has been demonstrated, the ecological significance of the rhizosphere-expressed TTSS of P. fluorescens SBW25 remains unclear.

Effect of opioid dependence pharmacotherapies on zidovudine disposition.

Injection drug users are frequently infected with human immunodeficiency virus (HIV) and receive opioid dependence pharmacotherapies and zidovudine (ZDV), the latter as a component of highly active antiretroviral therapy. We previously reported that methadone substantially increases ZDV concentrations. We now report on oral ZDV pharmacokinetics in 52 subjects receiving the opioid dependence pharmacotherapies l-alpha-acetylmethadol LAAM, buprenorphine, or naltrexone, and 17 non-opioid-treated controls. Relative to the area under the time-concentration curve (AUC) of ZDV in control subjects, no statistically significant differences in ZDV AUC were observed in participants treated with LAAM (p = .75), buprenorphine (p = .37), or naltrexone (p = .34). While methadone maintenance may result in ZDV toxicity and possibly require dose adjustments, other opioid pharmacotherapies should not produce ZDV toxicity.

Cue-dose training with monetary reinforcement: pilot study of an antiretroviral adherence intervention.

OBJECTIVE: To assess the feasibility and efficacy of two interventions for improving adherence to antiretroviral therapy regimens in HIV-infected subjects compared with a control intervention. DESIGN: Randomized, controlled, pilot study. SETTING: Department of Veterans Affairs HIV clinic and community-based HIV clinical trials site. PARTICIPANTS: Fifty-five HIV-infected subjects on stable antiretroviral therapy regimens. Subjects were predominantly male (89%) and African American (69%), and had histories of heroin or cocaine use (80%). INTERVENTIONS: Four weekly sessions of either nondirective inquiries about adherence (control group, C), cue-dose training, which consisted of the use of personalized cues for remembering particular dose times, and feedback about medication taking using Medication Event Monitoring System (MEMS) pill bottle caps, which record time of bottle opening (CD group), or cue-dose training combined with cash reinforcement for correctly timed bottle opening (CD+CR). MEASUREMENTS: Opening of the pill bottle within 2 hours before or after a predetermined time was measured by MEMS. RESULTS: Adherence to the medication as documented by MEMS was significantly enhanced during the 4-week training period in the CD+CR group, but not in the CD group, compared with the control group. Improvement was also seen in adherence to antiretroviral drugs that were not the object of training and reinforcement. Eight weeks after training and reinforcement were discontinued, adherence in the cash-reinforced group returned to near-baseline levels. CONCLUSIONS: Cue-dose training with cash reinforcement led to transient improvement in adherence to antiretroviral therapy in a population including mostly African Americans and subjects with histories of drug abuse. However, we were not able to detect any sustained improvement beyond the active training period, and questions concerning the timing and duration of such an intervention require further study. Randomized, controlled clinical studies with objective measures of adherence can be conducted in HIV-infected subjects and should be employed for further evaluation of this and other adherence interventions.

In vivo expression technology strategies: valuable tools for biotechnology.

Whole genome sequences have shown that bacteria possess a significant number of genes that have no known function. It is probable that many of these are required for survival in environments other than the agar plate. In vivo selection strategies provide a means of obtaining genes active in complex natural environments. Direct access to these genes is essential for understanding ecological performance and provides novel opportunities for biotechnology.

Interaction of methadone with didanosine and stavudine.

For opiate-dependent injection drug users infected with HIV, methadone therapy may facilitate adherence to complex highly active antiretroviral therapy (HAART) regimens. Current HAART regimens include one or more nucleoside analogues. We investigated the effects of methadone on the pharmacokinetics of the tablet formulation of didanosine (ddI) and of stavudine (d4T) in 17 study subjects on stable methadone therapy and in 10 untreated controls. Methadone treatment reduced the measured areas under the time-concentration curve (AUC0-6) by 63% for ddI (p =.04) and by 25% for d4T (p =.005) and the extrapolated AUCs for the full dosing interval (AUC0-12) by 57% for ddI (p =.11) and by 23% for d4T (p =. 02). Peak drug concentrations (Cmax) were reduced by 66% (p =.007) and 44% (p =.001) for ddI and d4T, respectively. The effects on AUC and Cmax appeared to result primarily from decreases in bioavailability. Methadone also delayed drug absorption. Trough levels for methadone did not differ significantly from those in historical controls, suggesting that ddI and d4T did not substantially alter methadone disposition. The results suggest that larger doses of the tablet formulation or an alternate formulation may be needed when didanosine is given to study subjects treated with methadone.

Diversity peaks at intermediate productivity in a laboratory microcosm.

The species diversity of natural communities is often strongly related to their productivity. The pattern of this relationship seems to vary: diversity is known to increase monotonically with productivity, to decrease monotonically with productivity, and to be unimodally related to productivity, with maximum diversity occurring at intermediate levels of productivity. The mechanism underlying these patterns remains obscure, although many possibilities have been suggested. Here we outline a simple mechanism--involving selection in a heterogeneous environment--to explain these patterns, and test it using laboratory cultures of the bacterium Pseudomonas fluorescens. We grew diverse cultures over a wide range of nutrient concentrations, and found a strongly unimodal relationship between diversity and productivity in heterogeneous, but not in homogeneous, environments. Our result provides experimental evidence that the unimodal relationship often observed in natural communities can be caused by selection for specialized types in a heterogeneous environment.

Disturbance and diversity in experimental microcosms.

External agents of mortality (disturbances) occur over a wide range of scales of space and time, and are believed to have large effects on species diversity. The "intermediate disturbance hypothesis", which proposes maximum diversity at intermediate frequencies of disturbance, has received support from both field and laboratory studies. Coexistence of species at intermediate frequencies of disturbance is thought to require trade-offs between competitive ability and disturbance tolerance, and a metapopulation structure, with disturbance affecting only a few patches at any given time. However, a unimodal relationship can also be generated by global disturbances that affect all patches simultaneously, provided that the environment contains spatial niches to which different species are adapted. Here we report the results of tests of this model using both isogenic and diverse populations of the bacterium Pseudomonas fluorescens. In both cases, a unimodal relationship between diversity and disturbance frequency was generated in heterogeneous, but not in homogeneous, environments. The cause of this relationship is competition among niche-specialist genotypes, which maintains diversity at intermediate disturbance, but not at high or low disturbance. Our results show that disturbance can modulate the effect of spatial heterogeneity on biological diversity in natural environments.

Solution and solid-state structures of phosphine adducts of monomeric zinc bisphenoxide complexes. Importance of these derivatives in CO2/epoxide copolymerization processes.

Phosphine derivatives of the monomeric zinc phenoxide complexes, (phenoxide)2ZnLn, where phenoxide equals 2,6-di-tert-butylphenoxide, 2,4,6-tri-tert-butylphenoxide, and 2,6-diphenylphenoxide and n = 1 or 2, have been synthesized from the reaction of Zn[N(SiMe3)2]2 and the corresponding phenol followed by the addition of phosphine. The complexes have been characterized in solution by 31P NMR spectroscopy and in selected instances in the solid-state by X-ray crystallography. The small, basic phosphine, PMe3, provided the only case of an isolated complex possessing two phosphine ligands (i.e., n = 2). For all other larger phosphines only the monophosphine adducts were obtained. Furthermore, only fairly basic phosphines were found to bind to zinc, e.g., whereas PPh3 (pKa = 2.73) was ineffective, PPh2Me (pKa = 4.57) did form a strong bond to zinc. The solid-state structures of the monophosphine adducts consist of a near-trigonal planar geometry about the zinc center, where the average P-Zn-O angles are larger than the O-Zn-O angles. On the other hand, the bisphosphine adduct, Zn(O-2,4,6-tBu3C6H2)(2).2PMe3, is a distorted tetrahedral structure with O-Zn-O and P-Zn-P bond angles of 108.8(2) degrees and 107.1(9) degrees, respectively. Competitive phosphine binding studies monitored by 31P NMR spectroscopy provided a relative binding order of PPh3 approximately PtBu3 << PPh2Me < PCy3 < PMe2Ph < PnBu3 < PEt3 < PMe3. Hence, the relative binding of basic phosphine ligands at these congested zinc sites is largely determined by their steric requirements. All phosphine adducts, with the exception of PMe2Ph and PMe3, were found to undergo slow self-exchange (< 600 s-1) with free phosphine by 31P NMR spectroscopy. However, the two small phosphines, PMe2Ph (cone angle = 122 degrees) and PMe3 (cone angle = 118 degrees), were shown to undergo rapid exchange presumably via an associative mechanism. Although there was no kinetic preferences for PCy3 binding to cadmium vs zinc, cadmium was thermodynamically favored by about a factor of 2.5. The addition of up to 3 equiv of PCy3 to the Zn(O-2,6-tBu2C6H3)2 or Zn(O-2,4,6-tBu3C6H2)2 derivatives did not significantly alter the reactivity of these catalysts for the copolymerization of cyclohexene oxide (CHO) and CO2 to high-molecular weight poly(cyclohexene carbonate). However, the presence of PCy3 greatly retarded their ability to homopolymerize CHO to polyether or to afford polyether linkages during the copolymerization of CHO/CO2.

Environmentally constrained mutation and adaptive evolution in Salmonella.

The relationship between environment and mutation is complex [1]. Claims of Lamarkian mutation [2] have proved unfounded [3-5]; it is apparent, however, that the external environment can influence the generation of heritable variation, through either direct effects on DNA sequence [6] or DNA maintenance and copying mechanisms [7-10], or as a consequence of evolutionary processes [11-16]. The spectrum of mutational events subject to environmental influence is unknown [6] and precisely how environmental signals modulate mutation is unclear. Evidence from bacteria suggests that a transient recombination-dependent hypermutational state can be induced by starvation [5]. It is also apparent that changes in the mutability of specific loci can be influenced by alterations in DNA topology [10,17]. Here we describe a remarkable instance of adaptive evolution in Salmonella which is caused by a mutation that occurs in intermediate-strength osmotic environments. We show that the mutation is not 'directed' and describe its genetic basis. We also present compelling evidence in support of the hypothesis that the mutational event is constrained by signals transmitted from the external environment via changes in the activity of DNA gyrase.

Performance characteristics of three serum iron and total iron-binding capacity methods in acute iron overdose.

Accurate serum iron and total iron binding capacity (TIBC) measurements may be useful in acute iron overdoses. Two alumina column TIBC methods were found to measure increased TIBC when free iron was present. A homogeneous TIBC method gave consistent results until iron concentrations exceeded 500 micrograms/dL (90 mumol/L), when it began to underestimate the TIBC. Serious iron overdoses require chelation therapy with deferoxamine. Iron recovery was reduced by up to 50% for all 3 methods with clinically achievable concentrations of deferoxamine 8,400 micrograms/dL (150 mumol/L). TIBC measurements by both alumina column methods were reduced by deferoxamine in the presence of free iron and unaffected when the iron concentration was less than the TIBC. The homogeneous TIBC method yielded falsely elevated results in the presence of free deferoxamine. Procedures that measure TIBC by addition of excess ferric iron followed by alumina adsorption are not suitable for monitoring TIBC in acute iron overdose. The homogeneous TIBC assay can be used in acute iron overdose but underestimates TIBC when iron concentrations exceed 500 micrograms/dL (90 mumol/L). None of the methods examined are useful for measuring iron or TIBC in the presence of deferoxamine.