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Rhizosphere colonizing plant growth promoting bacteria (PGPB) increase their competitiveness by producing diffusible toxic secondary metabolites, which inhibit competitors and deter predators. Many PGPB also have one or more Type VI Secretion System (T6SS), for the delivery of weapons directly into prokaryotic and eukaryotic cells. Studied predominantly in human and plant pathogens as a virulence mechanism for the delivery of effector proteins, the function of T6SS for PGPB in the rhizosphere niche is poorly understood. We utilized a collection of Pseudomonas chlororaphis 30–84 mutants deficient in one or both of its two T6SS and/or secondary metabolite production to examine the relative importance of each T6SS in rhizosphere competence, bacterial competition, and protection from bacterivores. A mutant deficient in both T6SS was less persistent than wild type in the rhizosphere. Both T6SS contributed to competitiveness against other PGPB or plant pathogenic strains not affected by secondary metabolite production, but only T6SS-2 was effective against strains lacking their own T6SS. Having at least one T6SS was also essential for protection from predation by several eukaryotic bacterivores. In contrast to diffusible weapons that may not be produced at low cell density, T6SS afford rhizobacteria an additional, more immediate line of defense against competitors and predators.

LuxR solos are common in plant-associated bacteria and increasingly recognized for playing important roles in plant-microbe interkingdom signaling. Unlike the LuxR-type transcriptional regulators of prototype LuxR/LuxI quorum sensing systems, luxR solos do not have a LuxI-type autoinducer synthase gene associated with them. LuxR solos in plant-pathogenic bacteria are important for virulence and in plant endosymbionts contribute to symbiosis. In the present study, we characterized an atypical LuxR solo, PcsR2, in the biological control species Pseudomonas chlororaphis 30–84 that is highly conserved among sequenced P. chlororaphis strains. Unlike most LuxR solos in the plant-associated bacteria characterized to date, pcsR2 is not associated with a proline iminopeptidase gene and the protein has an atypical N-terminal binding domain. We created a pcsR2 deletion mutant and used quantitative RT-PCR to show that the expression of pcsR2 and genes in the operon immediately downstream was upregulated ∼10-fold when the wild type strain was grown on wheat roots compared to planktonic culture. PcsR2 was involved in upregulation. Using a GFP transcriptional reporter, we found that expression of pcsR2 responded specifically to root-derived substrates as compared to leaf-derived substrates but not to endogenous AHLs. Compared to the wild type, the mutant was impaired in the ability to utilize root carbon and nitrogen sources in wheat root macerate and to colonize wheat roots. Phenazine production and most biofilm traits previously shown to be correlated with phenazine production also were diminished in the mutant. Gene expression of several of the proteins in the phenazine regulatory network including PhzR, Pip (phenazine inducing protein) and RpeA/RpeB were reduced in the mutant, and overexpression of these genes in trans restored phenazine production in the mutant to wild-type levels, indicating PcsR2 affects the activity of the these regulatory genes upstream of RpeA/RpeB via an undetermined mechanism. Our results indicate PcsR2 upregulates the expression of the adjacent operon in response to unknown wheat root-derived signals and belongs to a novel subfamily of LuxR-type transcriptional regulators found in sequenced P. chlororaphis strains.

Introduction One of the Sustainable Development Goals is to reduce the amount of maternal deaths. This study is essential in Brazil. Methods Epidemiological descriptive study regarding maternal deaths between 10 to 19 years old, according to race, age group, marital status, year of occurrence, schooling status, and ICD-10 category, during 2007 to 2017 in Brazil. Data was obtained from DATASUS. Results In the period, there were 2,767 maternal deaths between 10 to 19 years old; 152(5,5%) of them between 42 days to one year postpartum. Regarding age, 2,582(93%) were between 15 to 19 years old, 185(6,6%) between 10 to 14 years old. Regarding race, 1,565(56,5%) were brown, 761(27,5%) were white, 236(8,5%) were black, 69(2,5%) were indigenous. Regarding marital status, 2,060(74,4%) were single, 240(8,7%) were married. Regarding the cause of death, 542(19,5%) were due to maternal diseases with labor and puerperium complications, 348(12,5%) due to eclampsia, 209(7,5%) due to puerperal infection, 152(5,5%) due to other causes of late maternal death, 150(5,4%) due to gestational hypertension, 115(4%) due to urinary tract infection. Regarding schooling status 1,062(38,3%) had 4 to 7 school years 809(29%) had 8 to 11 years, 207(7%) had 1 to 3 years, 44(1,6%) had no schooling. The total amount of maternal deaths and late maternal deaths, respectively, according to the year, were: 220 and 12(2007), 247 and 12(2008), 267 and 7(2009), 273 and 6(2010), 242 and 12(2011), 225 and 17(2012), 241 and 17(2013), 239 and 20(2014), 235 and 19(2015), 214 and 17(2016), and 212 and 13(2017). Conclusions The limited 0,03% reduction in the amount of maternal deaths in this age group between 2007 to 2017is worrisome. The deaths among mostly single girls with some schooling level reveals that the country needs to review its sexual education policies, and to enforce guidance regarding family planning, as well as enable the female youth to recognize and denounce sexual aggressions. Key messages Know the characteristics of adolescents who suffers with the risk of maternal death in Brazil, in order to create ways of reducing these risks. Alert to Brazilian government to take part in the awareness process and start investing in improving the specialized public health service and access to information for these women.

Application of plant growth promoting bacteria may induce plant salt stress tolerance, however the underpinning microbial and plant mechanisms remain poorly understood. In the present study, the specific role of phenazine production by rhizosphere-colonizing Pseudomonas in mediating the inhibitory effects of salinity on wheat seed germination and seedling growth in four different varieties was investigated using Pseudomonas chlororaphis 30-84 (wild type) and isogenic derivatives deficient or enhanced in phenazine production. The results showed that varieties differed in how they responded to the salt stress treatment and the benefits derived from colonization by P. chlororaphis 30-84. In all varieties, the salt stress treatment significantly reduced seed germination, and in seedlings, reduced relative water content, increased reactive oxygen species (ROS) levels in leaves, and in three of four varieties, reduced shoot and root production compared to the no salt stress treatment. Inoculation of seeds with Pseudomonas chlororaphis 30-84 wild type or derivatives promoted salt-stress tolerance in seedlings of the four commercial winter wheat varieties tested, but the salt-stress tolerance phenotype was not entirely due to phenazine production. For example, all P. chlororaphis derivatives (including the phenazine-producing mutant) significantly improved relative water content in two varieties, Iba and CV 1, for which the salt stress treatment had a large impact. Importantly, all P. chlororaphis derivatives enabled the salt inhibited wheat varieties studied to maintain above ground productivity in saline conditions. However, only phenazine-producing derivatives enhanced the shoot or root growth of seedlings of all varieties under nonsaline conditions. Notably, ROS accumulation was reduced, and antioxidant enzyme (catalase) activity enhanced in the leaves of seedlings grown in saline conditions that were seed-treated with phenazine-producing P. chlororaphis derivatives as compared to noninoculated seedlings. The results demonstrate the capacity of P. chlororaphis to improve salt tolerance in wheat seedlings by promoting plant growth and reducing osmotic stress and a role for bacterial phenazine production in reducing redox stress.

Introduction One of the Sustainable Development Goals is to promote mental health. It is then mandatory to know the death profile of this population and to develop public policies to reduce it. Methods Epidemiological descriptive study regarding the proportion of indigenous deaths according to the variables: age group, year of occurrence, gender, region, municipality and ICD-10 category, in the period 2008 to 2017 in Brazil, using data from the national database DATASUS. Results From 2008 to 2017, there were 5,489 indigenous deaths due to external causes in Brazil, with 1,107 (20.1%) due to self-caused lesions. The annual average was 110.7 cases per year: 100 in 2008, 95 in 2009, 93 in 2010, 97 in 2011, 90 in 2012, 113 in 2013, 117 in 2014, 132 in 2015, 120 in 2016, and 150 in 2017. Regarding causes, strangling/hanging/suffocation were 979 (88.4%), self-intoxication was 31 (2.8%) with other chemicals and 21 (1.8%) with pesticides. Regarding age group, 365 (32.9%) were between 15 to 19 years old, 325 (29.3%) between 20 to 29, 150 (13.5%) between 30 to 39, 131 (11.8%) between 10 to 14, and 136 (12.2%) in other age groups. Regarding gender, 807 (72.9%) were male, 299 (27.0%) were female, and 1 (0.09%) was ignored. Regarding region, the Northern Region had 575 (51.9%) cases and the Central-Western Region had 403 (36.4%) cases. The municipality of São Gabriel da Cachoeira was the one with the highest amount of cases: 132 (12.7%). Conclusions The 50% increase in the amount of deaths by suicide among indigenous people in the analyzed period reveals the infficacy of established public policies in Brazil. The number of deaths among the youth reveals the community trauma of exploration, violence and dignity loss. The Nothern and Central-Western regions had conflicts due to land demarcation and prospecting, which explain the high rates. The reduction of such tragedy demands capacitation of all the public spheres. Key messages Evaluate the aspects that promote it and to propose effective interventions in environments of difficult access and in a historically vulnerable population. Discuss some relevant characteristics among natives who are vulnerable to practicing self-made injuries, in order to make it possible to create new ways to protect this population.

Introduction In Brazil, suicide is an alarming and growing health problem among adolescents. The Sustainable Development Goals aims to promote mental health. Methods Descriptive epidemiological study regarding the proportion of deaths by suicide according to ICD-10 classification, gender, Brazilian region, place of occurrence, race and year in adolescents from 10 to 19 years old in the period of 2008 to 2017 in Brazil, through the national system of health data, DATASUS. Results From 2008 to 2017, there were 175,543 deaths in adolescents due to external causes in Brazil; voluntarily self-inflicted injuries were the second main cause with 8,028 (4.6%) deaths - Among them, 728 occurred in 2008, 672 in 2009, 706 in 2010, 733 in 2011, 792 in 2012, 786 in 2013, 814 in 2014, 854 in 2015, 897 in 2016, and 1047 in 2017. Cases of strangulation/hanging/suffocation were 5,429 (67.2%); self-poisoning by pesticides were 512 (6.5%); intentional self-harm caused by the firing of another firearm and an unspecified firearm were 456 (5.7%). The most affected gender was male, with 5,536 (69%) deaths; female was 2,492 (31%). Among regions, the Southeast Region had 2,238 (27.9%) death records, followed by the Northeast, with 2,128 (26.5%). The most reported place of occurrence was home, with 4,570 (56.9%) deaths, followed by hospital, with 1,627 (20.3%). Among races, brown people were 3,803 (47.4%) deaths, and whites were 3,023 (37.7%). Conclusions Suicide of adolescents is a growing tragedy, with an increase of 55% in the analyzed period. Expanding studies on the theme will favor better conduct and management by health professionals. The timely identification of adolescents with a suicidal tendency and its specialized follow-up may be tools to reduce this sad reality. Key messages The study of child and youth suicide is important to assess the aspects that promote it, propose effective interventions and evaluate, by monitoring, the problems caused at home. It elucidates a important topic to Brazilian public health: suicide among young people. Due to its prevalence, discussing it corroborates to develop new guidelines and conducts for managing patients.

Pseudomonas chlororaphis 30-84 is a biological control agent selected for its ability to suppress diseases caused by fungal pathogens. P. chlororaphis 30-84 produces three phenazines: phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine-1-carboxylic acid (2OHPCA) and a small amount of 2-hydroxy-phenazine (2OHPHZ), and these are required for fungal pathogen inhibition and wheat rhizosphere competence. The two, 2-hydroxy derivatives are produced from PCA via the activity of a phenazine-modifying enzyme encoded by phzO. In addition to the seven biosynthetic genes responsible for the production of PCA, many other Pseudomonas strains possess one or more modifying genes, which encode enzymes that act independently or together to convert PCA into other phenazine derivatives. In order to understand the fitness effects of producing different phenazines, we constructed isogenic derivatives of P. chlororaphis 30-84 that differed only in the type of phenazines produced. Altering the type of phenazines produced by P. chlororaphis 30-84 enhanced the spectrum of fungal pathogens inhibited and altered the degree of take-all disease suppression. These strains also differed in their ability to promote extracellular DNA release, which may contribute to the observed differences in the amount of biofilm produced. All derivatives were equally important for survival over repeated plant/harvest cycles, indicating that the type of phenazines produced is less important for persistence in the wheat rhizosphere than whether or not cells produce phenazines. These findings provide a better understanding of the effects of different phenazines on functions important for biological control activity with implications for applications that rely on introduced or native phenazine producing populations.

Inpatient hospital stays for patients with epilepsy represent a significant burden on patients and society. Identifying factors that contribute to such costs aides in developing effective strategies to address this burden. July admissions have been associated with higher rates of complications and worse outcomes, attributed to the presence of new physicians. This study aims to evaluate whether epilepsy patients admitted in July have higher preventable complication rates and mortality than during the rest of the year.Data was derived from the Nationwide Inpatient Sample (NIS) for epilepsy admissions for the years 2000-2010. Multivariable analyses assessed the effect of July against non-July admission on "hospital acquired complications" (HAC), which are complications identified as owing to preventable causes and mortality. Additionally, the total adjusted charges and prolonged length of stay (pLOS) for July admissions were compared to the 50th percentile.A total of 12,997,181 admissions for epilepsy were identified with 993,619 (8%) occurring in July, 10,810,900 (83%) were non-July months, and 1,192,662 (9%) were missing data. Patients admitted in July showed an increased association for HAC events (RR=1.02, [1.01,1.03], p0.01), but a decrease in mortality (RR=0.96, [0.95,0.97], p0.01). There was no difference in rates of higher total adjusted charges for July admissions (RR=1.00, [1.00,1.00], p0.01) and a decrease in rates of pLOS (RR=0.99, [0.98,0.99], p0.01).In the epilepsy population, although July admissions were associated with a slight increase in HAC events, there was a non-significant or decreased rate of mortality, LOS, and total charge. Our results suggest that although complications were increased in July, possibly due to new staff, supervision is sufficient to prevent significant burden on patients and hospitals.

We describe the design and assembly of the LUX-ZEPLIN experiment, a direct detection search for cosmic WIMP dark matter particles. The centerpiece of the experiment is a large liquid xenon time projection chamber sensitive to low energy nuclear recoils. Rejection of backgrounds is enhanced by a Xe skin veto detector and by a liquid scintillator Outer Detector loaded with gadolinium for efficient neutron capture and tagging. LZ is located in the Davis Cavern at the 4850' level of the Sanford Underground Research Facility in Lead, South Dakota, USA. We describe the major subsystems of the experiment and its key design features and requirements. info:eu-repo/semantics/publishedVersion

The specific role of phenazines produced by rhizosphere-colonizing Pseudomonas in mediating wheat seedling drought-stress tolerance and recovery from water deficit was investigated using Pseudomonas chlororaphis 30-84 and isogenic derivatives deficient or enhanced in phenazine production compared to wild type. Following a 7-day water deficit, seedlings that received no-inoculum or were colonized by the phenazine mutant wilted to collapse, whereas seedlings colonized by phenazine producers displayed less severe symptoms. After a 7-day recovery period, survival of seedlings colonized by phenazine-producing strains exceeded 80%, but was less than 60% for no-inoculum controls. A second 7-day water deficit reduced overall survival rates to less than 10% for no-inoculum control seedlings, whereas survival was ∼50% for seedlings colonized by phenazine-producers. The relative water content of seedlings colonized by phenazine-producers was 10–20% greater than for the no-inoculum controls at every stage of water deficit and recovery, resulting in higher recovery indices than observed for the no-inoculum controls. For 10-day water deficits causing the collapse of all seedlings, survival rates remained high for plants colonized by phenazine-producers, especially the enhanced phenazine producer (∼74%), relative to the no-inoculum control (∼25%). These observations indicate that seedlings colonized by the phenazine-producing strains suffered less from dehydration during water deficit and recovered better, potentially contributing to better resilience from a second drought/recovery cycle. Seedlings colonized by phenazine-producing strains invested more in root systems and produced 1.5 to 2 fold more root tips than seedlings colonized by the phenazine mutant or the no-inoculum controls when grown with or without water deficit. The results suggest that the presence of phenazine-producing bacteria in the rhizosphere provides wheat seedlings with a longer adjustment period resulting in greater drought-stress avoidance and resilience.

B cells are key contributors to chronic autoimmune pathology in multiple sclerosis (MS). Clonally related B cells exist in the cerebrospinal fluid (CSF), meninges, and CNS parenchyma of MS patients. We sought to investigate the presence of clonally related B cells over time by performing Ig heavy chain variable region repertoire sequencing on B cells from longitudinally collected blood and CSF samples of MS patients (n = 10). All patients were untreated at the time of the initial sampling; the majority (n = 7) were treated with immune-modulating therapies 1.2 (±0.3 SD) years later during the second sampling. We found clonal persistence of B cells in the CSF of 5 patients; these B cells were frequently Ig class-switched and CD27(+). Specific blood B cell subsets appear to provide input into CNS repertoires over time. We demonstrate complex patterns of clonal B cell persistence in CSF and blood, even in patients on immune-modulating therapy. Our findings support the concept that peripheral B cell activation and CNS-compartmentalized immune mechanisms can in part be therapy resistant.

Many products of secondary metabolism are activated by quorum sensing (QS), yet even at cell densities sufficient for QS, their production may be repressed under suboptimal growth conditions via mechanisms that still require elucidation. For many beneficial plant-associated bacteria, secondary metabolites such as phenazines are important for their competitive survival and plant-protective activities. Previous work established that phenazine biosynthesis in Pseudomonas chlororaphis 30-84 is regulated by the PhzR/PhzI QS system, which in turn is regulated by transcriptional regulator Pip, two-component system RpeA/RpeB and stationary phase/stress sigma factor RpoS. Disruption of MiaA, a tRNA modification enzyme, altered primary metabolism and growth leading to widespread effects on secondary metabolism, including reduced phenazine production and oxidative stress tolerance. Thus, the miaA mutant provided the opportunity to examine the regulation of phenazine production in response to altered metabolism and growth or stress tolerance. Despite the importance of MiaA for translation efficiency, the most significant effect of miaA disruption on phenazine production was the reduction in the transcription of phzR, phzI and pip, whereas neither the transcription nor translation of RpeB, a transcriptional regulator of pip, was affected. Constitutive expression of rpeB or pip in the miaA mutant completely restored phenazine production, but it resulted in further growth impairment. Constitutive expression of RpoS alleviated sensitivity to oxidative stress resulting from RpoS translation inefficiency in the miaA mutant, but it did not restore phenazine production. Our results support the model that cells curtail phenazine biosynthesis under suboptimal growth conditions via RpeB/Pip-mediated regulation of QS.

B-cells are key contributors to chronic autoimmune pathology in multiple sclerosis (MS). Clonally related B-cells exist in the cerebrospinal fluid (CSF), meninges, and central nervous system (CNS) parenchyma of MS patients. Longitudinally stable CSF oligoclonal band (OCB) antibody patterns suggest some local CNS B-cell persistence; however, the longitudinal B-cell dynamics within and between the CSF and blood remain unknown. We sought to address this by performing immunoglobulin heavy chain variable region repertoire sequencing on B-cells from longitudinally collected blood and CSF samples of MS patients (n=10). All patients were untreated at the time of the initial sampling; the majority (n=7) were treated with immune modulating therapies 1.2 (+/−0.3 SD) years later during the second sampling. We found clonal persistence of B-cells in the CSF of five patients; these B-cells were frequently immunoglobulin (Ig) class-switched and CD27+. We identified specific blood B-cell subsets that appear to provide input into CNS repertoires over time. We demonstrate complex patterns of clonal B-cell persistence in CSF and blood, even in patients on high-efficacy immune modulating therapy. Our findings support the concept that peripheral B-cell activation and CNS-compartmentalized immune mechanisms are in part therapy-resistant.

R-tailocins are high-molecular-weight bacteriocins resembling bacteriophage tails. Pseudomonas chlororaphis 30-84 is a plant growth-promoting rhizobacterial (PGPR) strain that produces two distinct R-tailocin particles with different killing spectra. The two R-tailocins have different evolutionary histories but are released by the same lysis cassette. A previous study showed that both tailocins are important for pairwise competition with susceptible rhizosphere-colonizing strains; however, the broader role of tailocins in competition with the native rhizosphere microbiome was not tested. Genomic analysis of the P. chlororaphis 30-84 R-tailocin gene cluster uncovered the presence of three tail fiber genes in the tailocin 2 genetic module that could potentially result in tailocin 2 particles having different tail fibers and thus a wider killing spectrum. In this study, the tail fibers were found to incorporate onto different tailocin 2 particles, each with a distinct killing spectrum. A loss of production of one or both tailocins resulted in decreased P. chlororaphis 30-84 persistence within the wheat rhizosphere when in competition with the native microflora but not bulk soil. The capacity to produce three different versions of a single tailocin, each having one of three different types of tail fibers, is a previously unreported mechanism that leads to a broader R-tailocin killing spectrum. This study also provides evidence for the function of R-tailocins in competition with rhizosphere microbiome communities but not in bulk soil. IMPORTANCE Although R-tailocin gene clusters typically encode one tail fiber protein, three tail fiber-resembling genes were identified in association with one of the two sets of R-tailocin genes within the tailocin cluster of P. chlororaphis 30-84 and other sequenced P. chlororaphis strain genomes. This study confirmed that P. chlororaphis 30-84 not only produces two distinct tailocins, but that one of them is produced with three different types of tail fibers. This is a previously unreported strategy to increase the breadth of strains targeted by an R-tailocin. Our finding that R-tailocins produced by a PGPR Pseudomonas strain enhanced its persistence within the wheat rhizosphere microbiome confirms that R-tailocin production contributes to the population dynamics of rhizobacterial communities.

The rhizosphere-colonizing bacterium Pseudomonas chlororaphis 30-84 is an effective biological control agent against take-all disease of wheat. In this study, we characterize a small-colony variant (SCV) isolated from a P. chlororaphis 30-84 biofilm. The SCV exhibited pleiotropic phenotypes, including small cell size, slow growth and motility, low levels of phenazine production, and increased biofilm formation and resistance to antimicrobials. To better understand the genetic alterations underlying these phenotypes, RNA and whole-genome sequencing analyses were conducted comparing an SCV to the wild-type strain. Of the genome's 5,971 genes, transcriptomic profiling indicated that 1,098 (18.4%) have undergone substantial reprograming of gene expression in the SCV. Whole-genome sequence analysis revealed multiple alterations in the SCV, including mutations in yfiR (cyclic-di-GMP production), fusA (elongation factor), and cyoE (heme synthesis) and a 70-kb deletion. Genetic analysis revealed that the yfiR locus plays a major role in controlling SCV phenotypes, including colony size, growth, motility, and biofilm formation. Moreover, a point mutation in the fusA gene contributed to kanamycin resistance. Interestingly, the SCV can partially switch back to wild-type morphologies under specific conditions. Our data also support the idea that phenotypic switching in P. chlororaphis is not due to simple genetic reversions but may involve multiple secondary mutations. The emergence of these highly adherent and antibiotic-resistant SCVs within the biofilm might play key roles in P. chlororaphis natural persistence.

A role of B cells in multiple sclerosis (MS) is well established, but there is limited understanding of their involvement during active disease. Here, we examined cerebrospinal fluid (CSF) and peripheral blood (PB) B cells in treatment-naive patients with MS or high-risk clinically isolated syndrome. Using flow cytometry, we found increased CSF lymphocytes with a disproportionate increase of B cells compared with T cells in patients with gadolinium-enhancing (Gd+) lesions on brain MRI. Ig gene heavy chain variable region (Ig-VH) repertoire sequencing of CSF and PB B cells revealed clonal relationships between intrathecal and peripheral B cell populations, which could be consistent with migration of B cells to and activation in the CNS in active MS. In addition, we found evidence for bystander immigration of B cells from the periphery, which could be supported by a CXCL13 gradient between CSF and blood. Understanding what triggers B cells to migrate and home to the CNS may ultimately aid in the rational selection of therapeutic strategies to limit progression in MS.

Phenazines are bacterial secondary metabolites and play important roles in the antagonistic activity of the biological control strain P. chlororaphis 30-84 against take-all disease of wheat. The expression of the P. chlororaphis 30-84 phenazine biosynthetic operon (phzXYFABCD) is dependent on the PhzR/PhzI quorum sensing system located immediately upstream of the biosynthetic operon as well as other regulatory systems including Gac/Rsm. Bioinformatic analysis of the sequence between the divergently oriented phzR and phzX promoters identified features within the 5'-untranslated region (5'-UTR) of phzX that are conserved only among 2OHPCA producing Pseudomonas. The conserved sequence features are potentially capable of producing secondary structures that negatively modulate one or both promoters. Transcriptional and translational fusion assays revealed that deletion of 90-bp of sequence at the 5'-UTR of phzX led to up to 4-fold greater expression of the reporters with the deletion compared to the controls, which indicated this sequence negatively modulates phenazine gene expression both transcriptionally and translationally. This 90-bp sequence was deleted from the P. chlororaphis 30-84 chromosome, resulting in 30-84Enh, which produces significantly more phenazine than the wild-type while retaining quorum sensing control. The transcriptional expression of phzR/phzI and amount of AHL signal produced by 30-84Enh also were significantly greater than for the wild-type, suggesting this 90-bp sequence also negatively affects expression of the quorum sensing genes. In addition, deletion of the 90-bp partially relieved RsmE-mediated translational repression, indicating a role for Gac/RsmE interaction. Compared to the wild-type, enhanced phenazine production by 30-84Enh resulted in improvement in fungal inhibition, biofilm formation, extracellular DNA release and suppression of take-all disease of wheat in soil without negative consequences on growth or rhizosphere persistence. This work provides greater insight into the regulation of phenazine biosynthesis with potential applications for improved biological control.

R-type tailocins are high-molecular-weight bacteriocins that resemble bacteriophage tails and are encoded within the genomes of many Pseudomonas species. In this study, analysis of the P. chlororaphis 30-84 R-tailocin gene cluster revealed that it contains the structural components to produce two R-tailocins of different ancestral origins. Two distinct R-tailocin populations differing in length were observed in UV-induced lysates of P. chlororaphis 30-84 via transmission electron microscopy. Mutants defective in the production of one or both R-tailocins demonstrated that the killing spectrum of each tailocin is limited to Pseudomonas species. The spectra of pseudomonads killed by the two R-tailocins differed, although a few Pseudomonas species were either killed by or insusceptible to both tailocins. Tailocin release was disrupted by deletion of the holin gene within the tailocin gene cluster, demonstrating that the lysis cassette is required for the release of both R-tailocins. The loss of functional tailocin production reduced the ability of P. chlororaphis 30-84 to compete with an R-tailocin-sensitive strain within biofilms and rhizosphere communities. Our study demonstrates that Pseudomonas species can produce more than one functional R-tailocin particle sharing the same lysis cassette but differing in their killing spectra. This study provides evidence for the role of R-tailocins as determinants of bacterial competition among plant-associated Pseudomonas in biofilms and the rhizosphere. IMPORTANCE Recent studies have identified R-tailocin gene clusters potentially encoding more than one R-tailocin within the genomes of plant-associated Pseudomonas but have not demonstrated that more than one particle is produced or the ecological significance of the production of multiple R-tailocins. This study demonstrates for the first time that Pseudomonas strains can produce two distinct R-tailocins with different killing spectra, both of which contribute to bacterial competition between rhizosphere-associated bacteria. These results provide new insight into the previously uncharacterized role of R-tailocin production by plant-associated Pseudomonas species in bacterial population dynamics within surface-attached biofilms and on roots.

The nasST operon encodes the transcriptional regulators of assimilatory nitrate reductase operons in phylogenetically diverse bacteria. NasT is a RNA-binding antiterminator and helps RNA polymerase read through the regulatory terminator sequences upstream of the structural genes. NasS senses nitrate and nitrite and regulates the activity of NasT through stoichiometric interaction. In this study, we analyzed the nasST sequence in Azotobacter vinelandii and revealed that the nasS and nasT genes overlap by 19 nucleotides. Our genetic analyses suggested that translational initiation of NasT was coupled with NasS translation, a regulatory mechanism that prevents overproduction of NasT. The significance of tight control of nasT expression was demonstrated in a nasT-overexpression strain, where expression of the assimilatory nitrate reductase operon was deregulated.

The GacS/GacA two-component regulatory system activates the production of secondary metabolites including phenazines crucial for biological control activity in Pseudomonas chlororaphis 30-84. To better understand the role of the Gac system on phenazine regulation, transcriptomic analyses were conducted by comparing the wild-type strain to a gacA mutant. RNA-seq analysis identified 771 genes under GacA control, including many novel genes. Consistent with previous findings, phenazine biosynthetic genes were significantly downregulated in a gacA mutant. The transcript abundances of phenazine regulatory genes such as phzI, phzR, iopA, iopB, rpoS, and pip also were reduced. Moreover, the transcript abundance of three noncoding RNAs (ncRNAs) including rsmX, rsmY, and rsmZ was significantly decreased by gacA mutation consistent with the presence of consensus GacA-binding sites associated with their promoters. Our results also demonstrated that constitutive expression of rsmZ from a non-gac regulated promoter resulted in complete restoration of N-acyl-homoserine lactone (AHL) and phenazine production as well as the expression of other gac-dependent secondary metabolites in gac mutants. The role of RsmA and RsmE in phenazine production also was investigated. Overexpression of rsmE, but not rsmA, resulted in decreased AHL and phenazine production in P. chlororaphis, and only a mutation in rsmE bypassed the requirement for GacA in phenazine gene expression. In contrast, constitutive expression of the phzI/phzR quorum sensing system did not rescue phenazine production in the gacA mutant, indicating the direct posttranscriptional control by Gac on the phenazine biosynthetic genes. On the basis of these results, we propose a model to illustrate the hierarchic role of phenazine regulators modulated by Gac in the control of phenazine production. The transcriptomic analysis also was used to identify additional genes regulated by GacA that may contribute to the biological control capability of strain 30-84.

Caves are relatively accessible subterranean habitats ideal for the study of subsurface microbial dynamics and metabolisms under oligotrophic, non-photosynthetic conditions. A 454-pyrotag analysis of the V6 region of the 16S rRNA gene was used to systematically evaluate the bacterial diversity of ten cave surfaces within Kartchner Caverns, a limestone cave. Results showed an average of 1,994 operational taxonomic units (97 % cutoff) per speleothem and a broad taxonomic diversity that included 21 phyla and 12 candidate phyla. Comparative analysis of speleothems within a single room of the cave revealed three distinct bacterial taxonomic profiles dominated by either Actinobacteria, Proteobacteria, or Acidobacteria. A gradient in observed species richness along the sampling transect revealed that the communities with lower diversity corresponded to those dominated by Actinobacteria while the more diverse communities were those dominated by Proteobacteria. A 16S rRNA gene clone library from one of the Actinobacteria-dominated speleothems identified clones with 99 % identity to chemoautotrophs and previously characterized oligotrophs, providing insights into potential energy dynamics supporting these communities. The robust analysis conducted for this study demonstrated a rich bacterial diversity on speleothem surfaces. Further, it was shown that seemingly comparable speleothems supported divergent phylogenetic profiles suggesting that these communities are very sensitive to subtle variations in nutritional inputs and environmental factors typifying speleothem surfaces in Kartchner Caverns.

RpeA is a two-component sensor protein that negatively controls biosynthesis of phenazines, which are required for biological control activity by Pseudomonas chlororaphis 30-84. In this study, we identified the cognate response regulator RpeB and investigated how RpeA and RpeB interact with the PhzR/PhzI quorum sensing system and other known regulatory genes to control phenazine production. Quantitative real-time PCR revealed that, in contrast with an rpeA mutant, expression of the phenazine biosynthetic genes as well as the pip and phzR genes were significantly reduced in an rpeB mutant, suggesting positive control of phenazines by RpeB. Complementation assays showed that overexpression of pip in trans rescued phenazine production in an rpeB mutant, whereas multiple copies of rpeB genes were unable to restore phenazine production in a pip or phzR mutant. These results indicate that RpeA and RpeB differentially regulate phenazine production and act upstream of Pip and PhzR in the phenazine regulatory network. The differential regulatory functions for RpeA and RpeB also affected the capacity of 30-84 for fungal inhibition. Based on these results, a model is proposed to illustrate the relationship of RpeA/RpeB to other regulatory genes controlling phenazine biosynthesis in P. chlororaphis 30-84, a regulatory hierarchy that may be conserved in other pseudomonads and may play a role in stress response.

Kartchner Caverns is an oligotrophic subterranean environment that hosts a wide diversity of actively growing calcite speleothems (secondary mineral deposits). In a previous study, we demonstrated that bacterial communities extracted from these surfaces are quite complex and vary between formations. In the current study, we evaluated the influence of several environmental variables on the superficial bacterial community structure of 10 active formations located in close proximity to one another in a small room of Kartchner Caverns State Park, Arizona, USA. Physical (color, dimensions) and chemical (elemental profile and organic carbon concentration) properties, as well as the DGGE-based bacterial community structure of the formations were analyzed. While elemental concentration was found to vary among the formations, the differences in the community structure could not be correlated with concentrations of either organic carbon or any of the elements evaluated. In contrast, the locations of formations with...

Bacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit plant or animal hosts. The GacS/GacA two-component regulatory system positively controls the expression of many of these beneficial external products in pseudomonad bacteria. Natural populations often contain variants with defective Gac systems that do not produce most external products. These mutants benefit from a decreased metabolic load but do not appear to displace the wild type in nature. How could natural selection maintain the wild type in the presence of a mutant with enhanced growth? One hypothesis is that Gac mutants are “cheaters” that do not contribute to the public good, favored within groups but selected against between groups, as groups containing more mutants lose access to ecologically important external products. An alternative hypothesis is that Gac mutants have a mutualistic interaction with the wild type, so that each variant benefits by the presence of the other. In the biocontrol bacterium Pseudomonas chlororaphis strain 30-84, Gac mutants do not produce phenazines, which suppress competitor growth and are critical for biofilm formation. Here, we test the predictions of these alternative hypotheses by quantifying interactions between the wild type and the phenazine- and biofilm-deficient Gac mutant within growing biofilms. We find evidence that the wild type and Gac mutants interact mutualistically in the biofilm context, whereas a phenazine-defective structural mutant does not. Our results suggest that the persistence of alternative Gac phenotypes may be due to the stabilizing role of local mutualistic interactions.

On plants, microbial populations interact with each other and their host through the actions of secreted metabolites. However, the combined action of diverse organisms and their different metabolites on plant health has yet to be fully appreciated. Here, the multifactorial nature of these interactions, at the organismal and molecular level, leading to the biological control of plant diseases is reviewed. To do so, we describe in detail the ecological significance of three different classes of secondary metabolites and discuss how they might contribute to biological control. Specifically, the roles of auxin, acetoin, and phenazines are considered, because they represent very different but important types of secondary metabolites. We also describe how studies of the global regulation of bacterial secondary metabolism have led to the discovery of new genes and phenotypes related to plant health promotion. In conclusion, we describe three avenues for future research that will help to integrate these complex and diverse observations into a more coherent synthesis of bacterially mediated biocontrol of plant diseases.

Information concerning the bacterial and archaeal communities present on calcite speleothems in carbonate caves is of interest because the activity of these microbes has been implicated as a potential biogenic component in the formation of secondary mineral deposits. In addition, these speleothems may harbor unique, previously unidentified microbes. The current study presents a comparative analysis of the superficial bacterial and archaeal community structure of multiple stalactites from two different cave formations located in close proximity to each other in a nonhuman-impacted area of Kartchner Caverns, Arizona, USA. PCR-denaturing gradient gel electrophoresis analysis (PCR-DGGE) revealed that microbial communities sampled from stalactites of a single speleothem are more similar to each other than to the communities sampled from stalactites of an adjacent speleothem, suggesting that both bacterial and archaeal communities are speleothem-specific. SR-XRD analysis confirmed that both speleothems sampled ...

Phenazines constitute a large group of nitrogen-containing heterocyclic compounds produced by a diverse range of bacteria. Both natural and synthetic phenazine derivatives are studied due their impacts on bacterial interactions and biotechnological processes. Phenazines serve as electron shuttles to alternate terminal acceptors, modify cellular redox states, act as cell signals that regulate patterns of gene expression, contribute to biofilm formation and architecture, and enhance bacterial survival. Phenazines have diverse effects on eukaryotic hosts and host tissues, including the modification of multiple host cellular responses. In plants, phenazines also may influence growth and elicit induced systemic resistance. Here, we discuss emerging evidence that phenazines play multiple roles for the producing organism and contribute to their behavior and ecological fitness.

Pseudomonas chlororaphis strain 30-84 is a plant-beneficial bacterium that is able to control take-all disease of wheat caused by the fungal pathogen Gaeumannomyces graminis var. tritici. The production of phenazines (PZs) by strain 30-84 is the primary mechanism of pathogen inhibition and contributes to the persistence of strain 30-84 in the rhizosphere. PZ production is regulated in part by the PhzR/PhzI quorum-sensing (QS) system. Previous flow cell analyses demonstrated that QS and PZs are involved in biofilm formation in P. chlororaphis (V. S. R. K. Maddula, Z. Zhang, E. A. Pierson, and L. S. Pierson III, Microb. Ecol. 52:289-301, 2006). P. chlororaphis produces mainly two PZs, phenazine-1-carboxylic acid (PCA) and 2-hydroxy-PCA (2-OH-PCA). In the present study, we examined the effect of altering the ratio of PZs produced by P. chlororaphis on biofilm formation and pathogen inhibition. As part of this study, we generated derivatives of strain 30-84 that produced only PCA or overproduced 2-OH-PCA. Using flow cell assays, we found that these PZ-altered derivatives of strain 30-84 differed from the wild type in initial attachment, mature biofilm architecture, and dispersal from biofilms. For example, increased 2-OH-PCA production promoted initial attachment and altered the three-dimensional structure of the mature biofilm relative to the wild type. Additionally, both alterations promoted thicker biofilm development and lowered dispersal rates compared to the wild type. The PZ-altered derivatives of strain 30-84 also differed in their ability to inhibit the fungal pathogen G. graminis var. tritici. Loss of 2-OH-PCA resulted in a significant reduction in the inhibition of G. graminis var. tritici. Our findings suggest that alterations in the ratios of antibiotic secondary metabolites synthesized by an organism may have complex and wide-ranging effects on its biology.

Enhanced production of 2-hydroxy-phenazine-1-carboxylic acid (2-OH-PCA) by the biological control strain Pseudomonas chlororaphis 30–84 derivative 30-84O* was shown previously to promote cell adhesion and alter the three-dimensional structure of surfaceattached biofilms compared to the wild type. The current study demonstrates that production of 2-OH-PCA promotes the release of extracellular DNA, which is correlated with the production of structured biofilm matrix. Moreover, the essential role of the extracellular DNA in maintaining the mass and structure of the 30–84 biofilm matrix is demonstrated. To better understand the role of different phenazines in biofilm matrix production and gene expression, transcriptomic analyses were conducted comparing gene expression patterns of populations of wild type, 30-84O* and a derivative of 30–84 producing only PCA (30-84PCA) to a phenazine defective mutant (30-84ZN) when grown in static cultures. RNA-Seq analyses identified a group of 802 genes that were differentially expressed by the phenazine producing derivatives compared to 30-84ZN, including 240 genes shared by the two 2-OH-PCA producing derivatives, the wild type and 30-84O*. A gene cluster encoding a bacteriophage- derived pyocin and its lysis cassette was upregulated in 2-OH-PCA producing derivatives. A holin encoded in this gene cluster was found to contribute to the release of eDNA inmore » 30–84 biofilm matrices, demonstrating that the influence of 2-OH-PCA on eDNA production is due in part to cell autolysis as a result of pyocin production and release. The results expand the current understanding of the functions different phenazines play in the survival of bacteria in biofilm-forming communities.« less

BACKGROUND To evaluate the toxicity and pharmacological and biological properties of the farnesyl protein transferase (FPTase) inhibitor, tipifarnib (R115777, ZARNESTRAtrade mark) and capecitabine administered for 14 days every 3 weeks. PATIENTS AND METHODS Patients with advanced cancers received twice daily tipifarnib (100-500 mg) and capecitabine (1000-1125 mg/m(2)) for 14 days every 3 weeks. Pharmacokinetics of tipifarnib, capecitabine and 5-fluorouracil (5-FU) were determined. Peripheral blood mononuclear cells were analyzed for farnesylation of the HDJ2 chaperone protein and FPTase activity. RESULTS Forty-one patients received 185 courses of treatment. Diarrhea and palmar-plantar erythrodysesthesia were dose limiting at 300 mg tipifarnib/1125 mg/m(2) capecitabine b.i.d. When the capecitabine dose was fixed at 1000 mg/m(2) b.i.d., neutropenia was dose limiting at 400 and 500 mg b.i.d. of tipifarnib. Capecitabine did not affect the pharmacology of tipifarnib at 100-300 mg b.i.d., although tipifarnib significantly increased the C(max) of 5-FU at 400 mg b.i.d. HDJ2 farnesylation and FPTase activity decreased between 200 and 400 mg b.i.d. doses of tipifarnib, without a dose-response relationship. Five patients demonstrated partial remissions and 11 patients maintained prolonged stable disease. CONCLUSIONS Tipifarnib and capecitabine are well tolerated at 300 mg/1000 mg/m(2) b.i.d., respectively, resulting in biologically relevant plasma concentrations and antitumor activity. The recommended dose for further disease-focused studies is 300 mg b.i.d. tipifarnib and 1000 mg/m(2) b.i.d. capecitabine, given for 14 days every 3 weeks.

The biological control bacterium Pseudomonas chlororaphis (aureofaciens) strain 30-84 employs two quorum sensing (QS) systems: PhzR/PhzI regulates the production of the antibiotics phenazine-1-carboxylic acid, 2-hydroxy-phenazine-1-carboxylic acid, and 2-hydroxy-phenazine, whereas CsaR/CsaI regulates currently unknown aspects of the cell surface. Previously characterized derivatives of strain 30-84 with mutations in each QS system and in the phenazine biosynthetic genes were screened for their ability to form surface-attached biofilm populations in vitro, using microtiter plate and flow cell biofilm assays, and on seeds and roots. Results from in vitro, seed, and root studies demonstrated that the PhzR/PhzI and the CsaR/CsaI QS regulatory systems contribute to the establishment of biofilms, with mutations in PhzR/PhzI having a significantly greater effect than mutations in CsaR/CsaI. Interestingly, phenazine antibiotic production was necessary for biofilm formation to the same extent as the PhzR/PhzI QS system, suggesting the loss of phenazines was responsible for the majority of the biofilm defect in these mutants. In vitro analysis indicated that genetic complementation or AHL addition to the growth medium restored the ability of the AHL synthase phzI mutant to form biofilms. However, only phenazine addition or genetic complementation of the phenazine biosynthetic mutation in trans restored biofilm formation by mutants defective in the transcriptional activator phzR or the phzB structural mutant. QS and phenazine production were also involved in the establishment of surface-attached populations on wheat seeds and plant roots, and, as observed in vitro, the addition of AHL extracts restored the ability of phzI mutants, but not phzR mutants, to form surface attached populations on seeds. Similarly, the presence of the wild type in mixtures with the mutants restored the ability of the mutants to colonize wheat roots, demonstrating that AHL and/or phenazine production by the wild-type population could complement the AHL- and phenazine-deficient mutants in situ. Together, these data demonstrate that both QS systems are involved in the formation of surface-attached populations required for biofilm formation by P. chlororaphis strain 30-84, and indicate a new role for phenazine antibiotics in rhizosphere community development beyond inhibition of other plant-associated microorganisms.

SUMMARYRecent data from our laboratory suggest that sex steroids promote contractile function in cardiac muscle of rainbow trout (Oncorhynchus mykiss Walbaum), and there are sex differences in hormone signaling and cardiac function. The current study investigated whether steroid-induced inotropism in electrically paced (0.5 Hz, 14°C) ventricle strips at 90%Lmax (1) has a metabolic requirement for exogenous glucose and (2) is associated with enhanced intracellular Ca2+ storage and release from the sarcoplasmic reticulum (SR). We also explored whether sex differences exist in extracellular Ca2+(Ca2+o) or cardiac sensitivity to Ca2+o. In the absence or at low concentrations (1 or 2 mmol l-1) of exogenous glucose, resting tension and relaxation time were increased selectively in cardiac tissue from females. Increasing glucose promoted twitch force in a bell-shaped manner, with 5 mmol l-1representing the optimal concentration for both sexes. The positive inotropic effects of physiological concentrations of testosterone (T) and 17β-estradiol (E2) in male and female trout ventricle strips,respectively, developed slowly (10-45 min) and were not apparent in glucose-free medium, in medium containing iodoacetate (IAA), an inhibitor of glycolysis, or medium containing 5 mmol l-1 lactate or pyruvate. Male ventricle strips had increased inotropic responses to glucose and T compared with female strips exposed to glucose and E2. Furthermore, sexually maturing males showed a greater inotropic response than immature males or females. Pretreatment with ryanodine (a specific blocker of SR Ca2+release) also eliminated the inotropic effects of sex steroids and exogenous glucose and reduced the post-rest potentiation of contractile force (a marker of SR Ca2+ storage). By contrast, the inotropic effects of epinephrine (Epi) or elevated Ca2+o were faster(developing within 1-3 min) and were not diminished by the presence or absence of glucose or by pretreatment with IAA or ryanodine. Sex differences were also found in responsiveness to caffeine (males > females) and the relationship between Ca2+ concentration and force development above baseline. The Ca2+50 was lower in female cardiac tissue than males, suggesting greater Ca2+ sensitivity, and although plasma albumin was higher in females, total and ionized plasma Ca2+ did not differ between the sexes. For the first time, our study highlights the importance of extracellular glucose, glycolytic activity and SR Ca2+ storage and release for sex steroid-induced inotropism in the trout ventricle. Conversely, the inotropes Epi and elevated[Ca2+o] do not require the presence or metabolism of exogenous glucose or the SR for signaling their positive effects on contractility. These results also demonstrate novel sex-related differences in cardiac reliance on exogenous glucose, Ca2+ sensitivity and SR function and thus should be considered in future studies.

We analyzed changes in gene expression during male meiosis in Petunia by combining the meiotic staging of pollen mother cells from a single anther with cDNA-AFLP transcript profiling of mRNA from the synchronously developing sister anthers. The transcript profiling experiments focused on the identification of genes with a modulated expression profile during meiosis, while premeiotic archesporial cells and postmeiotic microspores served as a reference. About 8000 transcript tags, estimated at 30% of the total transcriptome, were generated, of which around 6% exhibited a modulated gene expression pattern at meiosis. Cluster analysis revealed a transcriptional cascade that coincides with the initiation and progression through all stages of the two meiotic divisions. Fragments that exhibited high expression specifically during meiosis I were characterized further by sequencing; 90 out of the 293 sequenced fragments showed homology with known genes, belonging to a wide range of gene classes, including previously characterized meiotic genes. In-situ hybridization experiments were performed to determine the spatial expression pattern for five selected transcript tags. Its concurrence with cDNA-AFLP transcript profiles indicates that this is an excellent approach to study genes involved in specialized processes such as meiosis. Our data set provides the potential to unravel unique meiotic genes that are as yet elusive to reverse genetics approaches.

Item does not contain fulltext The developmental expression pattern and localization of calreticulin were studied in Nicotiana tabacum L. anthers, pollen and pollen tubes. High transcript and protein levels were detected throughout anther development. Immunolocalization of calreticulin in the anthers showed particular dense label in tapetum and pollen at developmental stage 2, when the tapetum is highly active and the pollen tetrads are formed. Much lower transcript and protein levels were detected in dry and hydrated pollen and in pollen tubes. Immunofluorescence labeling of both chemically fixed and cryo-fixed and freeze-substituted pollen tubes showed the presence of calreticulin in Golgi apparatus and endoplasmic reticulum (ER). Calreticulin was seen throughout the stacks in the Golgi apparatus and in the areas with coated-Golgi vesicles but much less so in the ER. Calreticulin was not found in the secretory vesicles. A relatively intense label was occasionally seen adjacent to the wall of the tube. No significant label was observed in mitochondria, vacuoles, generative cells, cell wall or callose plugs. The present results are consistent with a role of calreticulin in Ca(2+)-dependent folding of secreted glycoproteins in tapetum, pollen and pollen tubes. 9 p.

Pseudomonas fluorescens Pf-5 is a plant commensal bacterium that inhabits the rhizosphere and produces secondary metabolites that suppress soilborne plant pathogens. The complete sequence of the 7.1-Mb Pf-5 genome was determined. We analyzed repeat sequences to identify genomic islands that, together with other approaches, suggested P. fluorescens Pf-5's recent lateral acquisitions include six secondary metabolite gene clusters, seven phage regions and a mobile genomic island. We identified various features that contribute to its commensal lifestyle on plants, including broad catabolic and transport capabilities for utilizing plant-derived compounds, the apparent ability to use a diversity of iron siderophores, detoxification systems to protect from oxidative stress, and the lack of a type III secretion system and toxins found in related pathogens. In addition to six known secondary metabolites produced by P. fluorescens Pf-5, three novel secondary metabolite biosynthesis gene clusters were also identified that may contribute to the biocontrol properties of P. fluorescens Pf-5. Supplementary information The online version of this article (doi:10.1038/nbt1110) contains supplementary material, which is available to authorized users.

Quorum sensing (QS) is a mechanism by which diverse microorganisms can control specific processes in response to population density. A relatively well-known form of QS among Proteobacteria involves production and subsequent response to acylated homoserine lactones (AHLs). Quorum sensing inhibition (QSI), targeting AHL-dependent signaling, has been reported as a strategy for the control of biofilm formation used by several marine organisms. We developed a simple soft agar overlay protocol, based on pigmentation inhibition, to rapidly screen for the presence of potential QSI by bacteria and plants. For bacterial screens, test organisms are first streaked onto their appropriate media and incubated overnight. For plant screens, the plant material (leaf, stem, flower, etc.) is placed onto LB agar. The bacterial growth or plant samples are then covered with an overlay of LB soft agar containing an inoculum of either Pseudomonas aureofaciens 30-84 or Chromobacterium violaceum ATCC 12472 (indicator cultures) and then incubated overnight. These indicator bacteria regulate pigment production by N-hexanoyl-HSL (C6-HSL) QS and are readily inhibited by AHL analogues and other antagonists. QSI is indicated by the lack of pigment production of the indicator culture in the vicinity of the test sample. Growth inhibition of the indicator culture indicates possible antibiotic production. Two different biosensor organisms based on derivatives of Agrobacterium tumefaciens and C. violaceum, capable of detecting a range of AHLs were used to determine whether QSI is due to the production of interfering AHLs competing with the C6-HSL regulation of C. violaceum and P. aureofaciens pigment production. This simple protocol will facilitate the screening of multiple organisms for the production of potential antifouling compounds.

Phenazine antibiotic production in the biological control bacterium Pseudomonas aureofaciens 30-84 is regulated in part via the PhzR/PhzI N -acyl homoserine lactone (AHL) system. Previous work showed that a subpopulation of the wheat rhizosphere community positively affected phenazine gene expression in strain 30-84 via AHL signals (E. A. Pierson, D. W. Wood, J. A. Cannon, F. M. Blachere, and L. S. Pierson III, Mol. Plant-Microbe Interact. 11: 1078-1084, 1998). In the present work, a second subpopulation, one that negatively affected phenazine gene expression, was identified from this rhizosphere community. Strain 30-84 grown in conditioned medium (CM) from several strains produced lower levels of phenazines (1.5- to 9.3-fold) than control when grown in CM from the strain 30-84I 1 /I 2 . Growth of the phzB :: lacZ reporter strain 30-84Z in this CM resulted in decreased lacZ expression (4.3- to 9.2-fold) compared to growth of the control strain in CM, indicating that inhibition of phzB occurred at the level of gene expression. Preliminary chemical and biological characterizations suggested that these signals, unlike other identified negative signals, were not extractable in ethyl acetate. Introduction of extra copies of phzR and phzI , but not phzI alone, in trans into strain 30-84Z reduced the negative effect on phzB :: lacZ expression. The presence of negative-signal-producing strains in a mixture with strain 30-84 reduced strain 30-84's ability to inhibit the take-all disease pathogen in vitro. Together, the results from the previous work on the positive-signal subpopulation and the present work on the negative-signal subpopulation suggest that cross-communication among members of the rhizosphere community and strain 30-84 may control secondary metabolite production and pathogen inhibition.

Bioassay-guided fractionation of a cytotoxic EtOAc extract of Aspergillus flavipes occurring in the rhizosphere of Ericameria laricifolia resulted in the isolation of three new cytochalasans, namely, aspochalasins I (1), J (2), and K (3), and four known cytochalasans, aspochalasins C (4), D (5), and E (6) and TMC-169 (7). The structures of compounds 1-3 were established on the basis of extensive 1D and 2D NMR spectroscopic analysis. All compounds exhibited weak to moderate cytotoxicity against NCI-H460, MCF-7, and SF-268 cancer cell lines, but none showed significant selectivity.

A novel cyclopentenedione, asterredione (1), two new terrecyclic acid A derivatives, (+)-5(6)-dihydro-6-methoxyterrecyclic acid A (2) and (+)-5(6)-dihydro-6-hydroxyterrecyclic acid A (3), and five known compounds, (+)-terrecyclic acid A (4), (-)-quadrone (5), betulinan A (6), asterriquinone D (7), and asterriquinone C-1 (8), were isolated from Aspergillus terreus occurring in the rhizosphere of Opuntia versicolor, using bioassay-guided fractionation. Acid-catalyzed reaction of 2 under mild conditions afforded 4, whereas under harsh conditions 2 yielded 5 and (-)-isoquadrone (9). Catalytic hydrogenation and methylation of 4 afforded 5(6)-dihydro-terrecyclic acid A (10) and (+)-terrecyclic acid A methyl ester (11), respectively. The structures of 1-11 were elucidated by spectroscopic methods. All compounds were evaluated for cytotoxicity in a panel of three sentinel cancer cell lines, NCI-H460 (non-small cell lung cancer), MCF-7 (breast cancer), and SF-268 (CNS glioma), and were found to be moderately active. Cell cycle analysis of 2, 4, and 5 using the NCI-H460 cell line indicated that 4 is capable of disrupting the cell cycle through an apparent arrest to progression at the G(1) and G(2)/M phases in this p53 competent cell line. A pathway for the biosynthetic origin of asterredione (1) from asterriquinone D (7) is proposed.

We investigated protracted low-dose oral Clofarabine for the treatment of myelodysplastic syndromes (MDS). Adults with an International Prognostic Scoring System (IPSS) score of INT-1 or higher who had failed first line therapy were eligible. INT-1 patients had to be transfusion-dependent. We started with oral Clofarabine at 5mg (fixed dose) daily for 10 consecutive days on a 28-day cycle. Toxicity prompted a modification to 1mg PO daily for 10 days and then 1mg PO daily for 7 days. Patients received treatment indefinitely until loss of response or unacceptable toxicity. Nine patients (5 women) were enrolled and evaluable (median age 65 years; range 55-81). A 10-day regimen of oral Clofarabine at 5mg/day induced Grade IV pancytopenia. A dose of 1 mg/day for 7/28 days was very well tolerated without significant toxicity. Three patients had responses (2 with responses lasting up to 21 and 51 cycles) defined as stable disease in spite of no significant change on bone marrow evaluation. Low-dose oral Clofarabine (1mg daily for 7/28 days) proved both effective and safe for patients with MDS who had failed prior therapy. This patient population is particularly sensitive to more protracted Clofarabine treatment schedules.

Environmental microorganisms normally exist as part of a community. In many cases, such communities are complex, and contain large numbers of diverse microbes that, in turn, result in natural ecosystems that provide a myriad of resources that benefit society. Fundamental to the success of these communities is their vast diversity which enables them to adapt to changing conditions. In this chapter we discuss key environmental diversity concepts including the questions of what is microbial diversity, what is a microbial community and what is a species? Subsequently, we discuss how environmental microorganisms interact with each other, with other organisms and with the environment. Community stability is also described including microbial megacommunities such as biofilms and rhizosphere communities. Finally, we discuss how diversity and interactions impact microbial adaptation, ecosystem function and the discovery of natural products.