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Ou Wang, , Graham Plastow, Kim Stanford, Brent Selinger,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01738-17

Abstract:
Cattle are the primary carrier of Escherichia coli O157:H7, a foodborne human pathogen, and those shedding >10 4 CFU/gram of feces of E. coli O157:H7 are defined as supershedders (SS). This study investigated the rectoanal junction (RAJ) mucosa-associated microbiota and its relationship with host gene expression in SS and in cattle from which E. coli O157:H7 was not detected (nonshedders [NS]), aiming to elucidate the mechanisms involved in supershedding. In total, 14 phyla, 66 families, and 101 genera of RAJ mucosa-associated bacteria were identified and Firmicutes (61.5 ± 7.5%), Bacteroidetes (27.9 ± 6.4%), and Proteobacteria (5.5 ± 2.1%) were the predominant phyla. Differential abundance analysis of operational taxonomic units (OTUs) identified 2 OTUs unique to SS which were members of Bacteroides and Clostridium and 7 OTUs unique to NS which were members of Coprococcus , Prevotella , Clostridium , and Paludibacter . Differential abundance analysis of predicted microbial functions (using PICRUSt [phylogenetic investigation of communities by reconstruction of unobserved states]) revealed that 3 pathways had higher abundance (log 2 fold change, 0.10 to 0.23) whereas 12 pathways had lower abundance (log 2 fold change, −0.36 to −0.20) in SS. In addition, we identified significant correlations between expression of 19 differentially expressed genes and the relative abundance of predicted microbial functions, including nucleic acid polymerization and carbohydrate and amino acid metabolism. Our findings suggest that differences in RAJ microbiota at both the compositional and functional levels may be associated with E. coli O157:H7 supershedding and that certain microbial groups and microbial functions may influence RAJ physiology of SS by affecting host gene expression. IMPORTANCE Cattle with fecal E. coli O157:H7 at >10 4 CFU per gram of feces have been defined as the supershedders, and they are responsible for the most of the E. coli O157:H7 spread into farm environment. Currently, no method is available for beef producers to eliminate shedding of E. coli O157:H7 in cattle, and the lack of information about the mechanisms of supershedding greatly impedes the development of effective methods. This study investigated the role of the rectoanal junction (RAJ) mucosa-associated microbiome in E. coli O157:H7 shedding, and our results indicated that the compositions and functions of RAJ microbiota differed between supershedders and nonshedders. The identified relationship between the differentially abundant microbes and 19 previously identified differentially expressed genes suggests the role of host-microbial interactions involved in E. coli O157:H7 supershedding. Our findings provide a fundamental understanding of the supershedding phenomenon which is essential for the development of strategies, such as the use of directly fed microbials, to reduce E. coli O157:H7 shedding in cattle.
Holly E. Saito, John R. Harp,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01633-17

Abstract:
Enterococcus faecalis is a commensal of the human gastrointestinal tract that can persist in the external environment and is a leading cause of hospital-acquired infections. Given its diverse habitats, the organism has developed numerous strategies to survive a multitude of environmental conditions. Previous studies have demonstrated that E. faecalis will incorporate fatty acids from bile and serum into its membrane, resulting in an induced tolerance to membrane-damaging agents. To discern whether all fatty acids induce membrane stress protection, we examined how E. faecalis responded to individually supplied fatty acids. E. faecalis readily incorporated fatty acids 14 to 18 carbons in length into its membrane but poorly incorporated fatty acids shorter or longer than this length. Supplementation with saturated fatty acids tended to increase generation time and lead to altered cellular morphology in most cases. Further, exogenously supplied saturated fatty acids did not induce tolerance to the membrane-damaging antibiotic daptomycin. Supplementation with unsaturated fatty acids produced variable growth effects, with some impacting generation time and morphology. Exogenously supplied unsaturated fatty acids that are normally produced by E. faecalis and those that are found in bile or serum could restore growth in the presence of a fatty acid biosynthetic inhibitor. However, only the eukaryote-derived fatty acids oleic acid and linoleic acid provided protection from daptomycin. Thus, exogenous fatty acids do not lead to a common physiological effect on E. faecalis . The organism responds uniquely to each, and only host-derived fatty acids induce membrane protection. IMPORTANCE Enterococcus faecalis is a commonly acquired hospital infectious agent with resistance to many antibiotics, including those that target its cellular membrane. We previously demonstrated that E. faecalis will incorporate fatty acids found in human fluids, like serum, into its cellular membrane, thereby altering its membrane composition. In turn, the organism is better able to survive membrane-damaging agents, including the antibiotic daptomycin. We examined fatty acids commonly found in serum and those normally produced by E. faecalis to determine which fatty acids can induce protection from membrane damage. Supplementation with individual fatty acids produced a myriad of different effects on cellular growth, morphology, and stress response. However, only host-derived unsaturated fatty acids provided stress protection. Future studies are aimed at understanding how these specific fatty acids induce protection from membrane damage.
Hao Yu, , Aijie Wang, Jianping Xie, Liyou Wu, Joy D. Van Nostrand, Decai Jin, Zhimin Shao, , Jizhong Zhou, et al.
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01694-17

Abstract:
Numerous studies have shown that the continuous increase of atmosphere CO 2 concentrations may have profound effects on the forest ecosystem and its functions. However, little is known about the response of belowground soil microbial communities under elevated atmospheric CO 2 (eCO 2 ) at different soil depth profiles in forest ecosystems. Here, we examined soil microbial communities at two soil depths (0 to 5 cm and 5 to 15 cm) after a 10-year eCO 2 exposure using a high-throughput functional gene microarray (GeoChip). The results showed that eCO 2 significantly shifted the compositions, including phylogenetic and functional gene structures, of soil microbial communities at both soil depths. Key functional genes, including those involved in carbon degradation and fixation, methane metabolism, denitrification, ammonification, and nitrogen fixation, were stimulated under eCO 2 at both soil depths, although the stimulation effect of eCO 2 on these functional markers was greater at the soil depth of 0 to 5 cm than of 5 to 15 cm. Moreover, a canonical correspondence analysis suggested that NO 3 -N, total nitrogen (TN), total carbon (TC), and leaf litter were significantly correlated with the composition of the whole microbial community. This study revealed a positive feedback of eCO 2 in forest soil microbial communities, which may provide new insight for a further understanding of forest ecosystem responses to global CO 2 increases. IMPORTANCE The concentration of atmospheric carbon dioxide (CO 2 ) has continuously been increasing since the industrial revolution. Understanding the response of soil microbial communities to elevated atmospheric CO 2 (eCO 2 ) is important for predicting the contribution of the forest ecosystem to global atmospheric change. This study analyzed the effect of eCO 2 on microbial communities at two soil depths (0 to 5 cm and 5 to 15 cm) in a forest ecosystem. Our findings suggest that the compositional and functional structures of microbial communities shifted under eCO 2 at both soil depths. More functional genes involved in carbon, nitrogen, and phosphorus cycling were stimulated under eCO 2 at the soil depth of 0 to 5 cm than at the depth of 5 to 15 cm.
, Angela Ruscitto,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01759-17

Abstract:
Tannerella forsythia and Fusobacterium nucleatum are dental plaque bacteria implicated in the development of periodontitis. These two species have been shown to form synergistic biofilms and have been found to be closely associated in dental plaque biofilms. A number of genetic loci for TonB-dependent membrane receptors (TDR) for glycan acquisition, with many existing in association with genes coding for enzymes involved in the breakdown of complex glycans, have been identified in T. forsythia . In this study, we focused on a locus, BFO_0186-BFO_0188, that codes for a predicted TDR-SusD transporter along with a putative β-glucan hydrolyzing enzyme (BFO_0186). This operon is located immediately downstream of a 2-gene operon that codes for a putative stress-responsive extracytoplasmic function (ECF) sigma factor and an anti-sigma factor. Here, we show that BFO_0186 expresses a β-glucanase that cleaves glucans with β-1,6 and β-1,3 linkages. Furthermore, the BFO_0186-BFO_0188 locus is upregulated, with an induction of β-glucanase activity, in cobiofilms of T. forsythia and F. nucleatum . The β-glucanase activity in mixed biofilms in turn leads to an enhanced hydrolysis of β-glucans and release of glucose monomers and oligomers as nutrients for F. nucleatum . In summary, our study highlights the role of T. forsythia β-glucanase expressed by the asaccharolytic oral bacterium T. forsythia in the development of T. forsythia-F. nucleatum mixed species biofilms, and suggest that dietary β-glucans might contribute in plaque development and periodontal disease pathogenesis. IMPORTANCE The development of dental plaque biofilm is a complex process in which metabolic, chemical and physical interactions between bacteria take a central role. Previous studies have shown that the dental pathogens T. forsythia and F. nucleatum form synergistic biofilms and are closely associated in human dental plaque. In this study, we show that β-glucanase from the periodontal pathogen T. forsythia plays a role in the formation of T. forsythia - F. nucleatum cobiofilms by hydrolyzing β-glucans to glucose as a nutrient. We also unveiled that the expression of T. forsythia β-glucanase is induced in response to F. nucleatum sensing. This study highlights the involvement of β-glucanase activity in the development of T. forsythia-F. nucleatum biofilms and suggests that intake of dietary β-glucans might be a contributing risk factor in plaque development and periodontal disease pathogenesis.
, Kenji Uehara, Yuichiro Tsukioka
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01845-17

Abstract:
In soy sauce manufacturing, Candida versatilis plays a role in the production of volatile flavor compounds, such as volatile phenols, but limited accessible information on its genome has prevented further investigation regarding aroma production and breeding. Although the draft genome sequence data of two strains of C. versatilis have recently been reported, these strains are not similar to each other. Here, we reassess the draft genome sequence data for strain t-1, which was originally reported to be C. versatilis , and conclude that strain t-1 is most probably not C. versatilis but a gamete of hybrid Zygosaccharomyces rouxii . Phylogenetic analysis of the D1/D2 region of the 26S ribosomal DNA (rDNA) sequence indicated that strain t-1 is more similar to the genus Zygosaccharomyces than to C. versatilis . Moreover, we found that the genome of strain t-1 is composed of haploid genome content and divided into two regions that show approximately 100% identity with the T or P subgenome derived from the natural hybrid Zygosaccharomyces rouxii , such as NBRC110957 and NBRC1876. We also found a chromosome crossing-over signature in the scaffolds of strain t-1. These results suggest that strain t-1 is a gamete of the hybrid Z. rouxii , generated by either meiosis or chromosome loss following reciprocal translocation between the T and P subgenomes. Although it is unclear why strain t-1 was misidentified as C. versatilis , the genome of strain t-1 has broad implications for considering the evolutionary fate of an allodiploid. IMPORTANCE In yeast, crossing between different species sometimes leads to interspecies hybrids. The hybrid generally cannot produce viable spores because dissimilarity of parental genomes prevents normal chromosome segregation during meiotic division, leading to a dead end. Thus, only a few natural cases of homoploid hybrid speciation, which requires mating between 1n gametes of hybrids, have been described. However, a recent study provided strong evidence that homoploid hybrid speciation is initiated in natural populations of the budding yeast, suggesting the potential presence of viable 1n gametes of hybrids. The significance of our study is finding that the strain t-1, which had been misidentified as Candida versatilis , is a viable 1n gamete derived from hybrid Zygosaccharomyces rouxii .
, Ivo Sedláček, Adéla Indráková, Veronika Vrbovská, Ivana Mašlaňová, Vojtěch Kovařovic, , , Lucie Krištofová, Jana Kekláková, et al.
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01746-17

Abstract:
Two Gram-stain-positive, coagulase-negative staphylococcal strains were isolated from abiotic sources comprising stone fragments and sandy soil in James Ross Island, Antarctica. Here, we describe properties of a novel species of the genus Staphylococcus that has a 16S rRNA gene sequence nearly identical to that of Staphylococcus saprophyticus . However, compared to S. saprophyticus and the next closest relatives, the new species demonstrates considerable phylogenetic distance at the whole-genome level, with an average nucleotide identity of <85% and inferred DNA-DNA hybridization of <30%. It forms a separate branch in the S. saprophyticus phylogenetic clade as confirmed by multilocus sequence analysis of six housekeeping genes, rpoB , hsp60 , tuf , dnaJ , gap , and sod . Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and key biochemical characteristics allowed these bacteria to be distinguished from their nearest phylogenetic neighbors. In contrast to S. saprophyticus subsp. saprophyticus , the novel strains are pyrrolidonyl arylamidase and β-glucuronidase positive and β-galactosidase negative, nitrate is reduced, and acid produced aerobically from d -mannose. Whole-genome sequencing of the 2.69-Mb large chromosome revealed the presence of a number of mobile genetic elements, including the 27-kb pseudo-staphylococcus cassette chromosome mec of strain P5085 T (ψSCC mecP5085 ), harboring the mecC gene, two composite phage-inducible chromosomal islands probably essential to adaptation to extreme environments, and one complete and one defective prophage. Both strains are resistant to penicillin G, ampicillin, ceftazidime, methicillin, cefoxitin, and fosfomycin. We hypothesize that antibiotic resistance might represent an evolutionary advantage against beta-lactam producers, which are common in a polar environment. Based on these results, a novel species of the genus Staphylococcus is described and named Staphylococcus edaphicus sp. nov. The type strain is P5085 T (= CCM 8730 T = DSM 104441 T ). IMPORTANCE The description of Staphylococcus edaphicus sp. nov. enables the comparison of multidrug-resistant staphylococci from human and veterinary sources evolved in the globalized world to their geographically distant relative from the extreme Antarctic environment. Although this new species was not exposed to the pressure of antibiotic treatment in human or veterinary practice, mobile genetic elements carrying antimicrobial resistance genes were found in the genome. The genomic characteristics presented here elucidate the evolutionary relationships in the Staphylococcus genus with a special focus on antimicrobial resistance, pathogenicity, and survival traits. Genes encoded on mobile genetic elements were arranged in unique combinations but retained conserved locations for the integration of mobile genetic elements. These findings point to enormous plasticity of the staphylococcal pangenome, shaped by horizontal gene transfer. Thus, S. edaphicus can act not only as a reservoir of antibiotic resistance in a natural environment but also as a mediator for the spread and evolution of resistance genes.
Yun Yang, Yichao Wu, Yidan Hu, Hua Wang, , James K. Fredrickson,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01693-17

Abstract:
Although biocatalytic transformation has shown great promise in chemical synthesis, there remain significant challenges in controlling high selectivity without the formation of undesirable by-products. For instance, few attempts to construct biocatalysts for de novo synthesis of pure flavin mononucleotide (FMN) have been successful, due to riboflavin (RF) accumulating in the cytoplasm and being secreted with FMN. To address this problem, we show here a novel biosynthesis strategy, compartmentalizing the final FMN biosynthesis step in the periplasm of an engineered Escherichia coli strain. This construct is able to overproduce FMN with high specificity (92.4% of total excreted flavins). Such a biosynthesis approach allows isolation of the final biosynthesis step from the cytoplasm to eliminate undesirable by-products, providing a new route to develop biocatalysts for the synthesis of high-purity chemicals. IMPORTANCE The periplasm of Gram-negative bacterial hosts is engineered to compartmentalize the final biosynthesis step from the cytoplasm. This strategy is promising for the overproduction of high-value products with high specificity. We demonstrate the successful implementation of this strategy in microbial production of highly pure FMN.
, Laurie Schönholzer-Mauclaire, Emmanuel Frossard,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01715-17

Abstract:
Soil and plant inoculation with heterotrophic zinc-solubilizing bacteria (ZSB) is considered a promising approach for increasing zinc (Zn) phytoavailability and enhancing crop growth and nutritional quality. Nevertheless, it is necessary to understand the underlying bacterial solubilization processes to predict their repeatability in inoculation strategies. Acidification via gluconic acid production remains the most reported process. In this study, wheat rhizosphere soil serial dilutions were plated on several solid microbiological media supplemented with scarcely soluble Zn oxide (ZnO), and 115 putative Zn-solubilizing isolates were directly detected based on the formation of solubilization halos around the colonies. Eight strains were selected based on their Zn solubilization efficiency and siderophore production capacity. These included one strain of Curtobacterium , two of Plantibacter , three strains of Pseudomonas , one of Stenotrophomonas , and one strain of Streptomyces . In ZnO liquid solubilization assays, the presence of glucose clearly stimulated organic acid production, leading to medium acidification and ZnO solubilization. While solubilization by Streptomyces and Curtobacterium was attributed to the accumulated production of six and seven different organic acids, respectively, the other strains solubilized Zn via gluconic, malonic, and oxalic acids exclusively. In contrast, in the absence of glucose, ZnO dissolution resulted from proton extrusion (e.g., via ammonia consumption by Plantibacter strains) and complexation processes (i.e., complexation with glutamic acid in cultures of Curtobacterium ). Therefore, while gluconic acid production was described as a major Zn solubilization mechanism in the literature, this study goes beyond and shows that solubilization mechanisms vary among ZSB and are strongly affected by growth conditions. IMPORTANCE Barriers toward a better understanding of the mechanisms underlying zinc (Zn) solubilization by bacteria include the lack of methodological tools for isolation, discrimination, and identification of such organisms. Our study proposes a direct bacterial isolation procedure, which prevents the need to screen numerous bacterial candidates (for which the ability to solubilize Zn is unknown) for recovering Zn-solubilizing bacteria (ZSB). Moreover, we confirm the potential of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) as a quick and accurate tool for the identification and discrimination of environmental bacterial isolates. This work also describes various Zn solubilization processes used by wheat rhizosphere bacteria, including proton extrusion and the production of different organic acids among bacterial strains. These processes were also clearly affected by growth conditions (i.e., solid versus liquid cultures and the presence and absence of glucose). Although highlighted mechanisms may have significant effects at the soil-plant interface, these should only be transposed cautiously to real ecological situations.
Daniel P. Kiesenhofer, ,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01742-17

Abstract:
Trichoderma reesei can produce up to 100 g/liter of extracellular proteins. The major and industrially relevant products are cellobiohydrolase I (CBHI) and the hemicellulase XYNI. The genes encoding both enzymes are transcriptionally activated by the regulatory protein Xyr1. The first 850 nucleotides of the cbh1 promoter contain 14 Xyr1-binding sites (XBS), and 8 XBS are present in the xyn1 promoter. Some of these XBS are arranged in tandem and others as inverted repeats. One such cis element, an inverted repeat, plays a crucial role in the inducibility of the xyn1 promoter. We investigated the impact of the properties of such cis elements by shuffling them by insertion, exchange, deletion, and rearrangement of cis elements in both the cbh1 and xyn1 promoter. A promoter-reporter assay using the Aspergillus niger goxA gene allowed us to measure changes in the promoter strength and inducibility. Most strikingly, we found that an inverted repeat of XBS causes an important increase in cbh1 promoter strength and allows induction by xylan or wheat straw. Furthermore, evidence is provided that the distances of cis elements to the transcription start site have important influence on promoter activity. Our results suggest that the arrangement and distances of cis elements have large impacts on the strength of the cbh1 promoter, whereas the sheer number of XBS has only secondary importance. Ultimately, the biotechnologically important cbh1 promoter can be improved by cis element rearrangement. IMPORTANCE In the present study, we demonstrate that the arrangement of cis elements has a major impact on promoter strength and inducibility. We discovered an influence on promoter activity by the distances of cis elements to the transcription start site. Furthermore, we found that the configuration of cis elements has a greater effect on promoter strength than does the sheer number of transactivator binding sites present in the promoter. Altogether, the arrangement of cis elements is an important factor that should not be overlooked when enhancement of gene expression is desired.
Pedro J. Cabello-Yeves, , , , Alexandra S. Zakharenko, Vadim V. Blinov,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.02132-17

Abstract:
We present a metagenomic study of Lake Baikal (East Siberia). Two samples obtained from the water column under the ice cover (5 and 20 m deep) in March 2016 have been deep sequenced and the reads assembled to generate metagenome-assembled genomes (MAGs) that are representative of the microbes living in this special environment. Compared with freshwater bodies studied around the world, Lake Baikal had an unusually high fraction of Verrucomicrobia . Other groups, such as Actinobacteria and Proteobacteria , were in proportions similar to those found in other lakes. The genomes (and probably cells) tended to be small, presumably reflecting the extremely oligotrophic and cold prevalent conditions. Baikal microbes are novel lineages recruiting very little from other water bodies and are distantly related to other freshwater microbes. Despite their novelty, they showed the closest relationship to genomes discovered by similar approaches from other freshwater lakes and reservoirs. Some of them were particularly similar to MAGs from the Baltic Sea, which, although it is brackish, connected to the ocean, and much more eutrophic, has similar climatological conditions. Many of the microbes contained rhodopsin genes, indicating that, in spite of the decreased light penetration allowed by the thick ice/snow cover, photoheterotrophy could be widespread in the water column, either because enough light penetrates or because the microbes are already adapted to the summer ice-less conditions. We have found a freshwater SAR11 subtype I/II representative showing striking synteny with Pelagibacter ubique strains, as well as a phage infecting the widespread freshwater bacterium Polynucleobacter . IMPORTANCE Despite the increasing number of metagenomic studies on different freshwater bodies, there is still a missing component in oligotrophic cold lakes suffering from long seasonal frozen cycles. Here, we describe microbial genomes from metagenomic assemblies that appear in the upper water column of Lake Baikal, the largest and deepest freshwater body on Earth. This lake is frozen from January to May, which generates conditions that include an inverted temperature gradient (colder up), decrease in light penetration due to ice, and, especially, snow cover, and oligotrophic conditions more similar to the open-ocean and high-altitude lakes than to other freshwater or brackish systems. As could be expected, most reconstructed genomes are novel lineages distantly related to others in cold environments, like the Baltic Sea and other freshwater lakes. Among them, there was a broad set of streamlined microbes with small genomes/intergenic spacers, including a new nonmarine Pelagibacter -like (subtype I/II) genome.
Da Liu, Yue Cui, ,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01888-17

Abstract:
Vegetable seeds contaminated with bacterial pathogens have been linked to fresh-produce-associated outbreaks of gastrointestinal infections. This study was undertaken to observe the physiological behavior of Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC) cells artificially internalized into vegetable seeds during the germination process. Surface-decontaminated seeds of alfalfa, fenugreek, lettuce, and tomato were vacuum-infiltrated with four individual strains of Salmonella or EHEC. Contaminated seeds were germinated at 25°C for 9 days, and different sprout/seedling tissues were microbiologically analyzed every other day. The internalization of Salmonella and EHEC cells into vegetable seeds was confirmed by the absence of pathogens in seed-rinsing water and the presence of pathogens in seed homogenates after postinternalization seed surface decontamination. Results show that 317 (62%) and 343 (67%) of the 512 collected sprout/seedling tissue samples were positive for Salmonella and EHEC, respectively. The average Salmonella populations were significantly larger ( P< 0.05) than the EHEC populations. Significantly larger Salmonella populations were recovered from the cotyledon and seed coat tissues, followed by the root tissues, but the mean EHEC populations from all sampled tissue sections were statistically similar, except in pregerminated seeds. Three Salmonella and two EHEC strains had significantly larger cell populations on sprout/seedling tissues than other strains used in the study. Salmonella and EHEC populations from fenugreek and alfalfa tissues were significantly larger than those from tomato and lettuce tissues. The study showed the fate of internalized human pathogens on germinating vegetable seeds and sprout/seedling tissues and emphasized the importance of using pathogen-free seeds for sprout production. IMPORTANCE The internalization of microorganisms into vegetable seeds could occur naturally and represents a possible pathway of vegetable seed contamination by human pathogens. The present study investigated the ability of two important bacterial pathogens, Salmonella and enterohemorrhagic Escherichia coli (EHEC), when artificially internalized into vegetable seeds, to grow and disseminate along vegetable sprouts/seedlings during germination. The data from the study revealed that the pathogen cells artificially internalized into vegetable seeds caused the contamination of different tissues of sprouts/seedlings and that pathogen growth on germinating seeds is bacterial species and vegetable seed-type dependent. These results further stress the necessity of using pathogen-free vegetable seeds for edible sprout production.
, Jason R. Westrich, Magdy S. Alabady, Martinique L. Edwards,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01035-17

Abstract:
The critically endangered elkhorn coral ( Acropora palmata ) is affected by white pox disease (WPX) throughout the Florida Reef Tract and wider Caribbean. The bacterium Serratia marcescens was previously identified as one etiologic agent of WPX but is no longer consistently detected in contemporary outbreaks. It is now believed that multiple etiologic agents cause WPX; however, to date, no other potential pathogens have been thoroughly investigated. This study examined the association of Vibrio bacteria with WPX occurrence from August 2012 to 2014 at Looe Key Reef in the Florida Keys, USA. The concentration of cultivable Vibrio was consistently greater in WPX samples than in healthy samples. The abundance of Vibrio bacteria relative to total bacteria was four times higher in samples from WPX lesions than in adjacent apparently healthy regions of diseased corals based on quantitative PCR (qPCR). Multilocus sequence analysis (MLSA) was used to assess the diversity of 69 Vibrio isolates collected from diseased and apparently healthy A. palmata colonies and the surrounding seawater. Vibrio species with known pathogenicity to corals were detected in both apparently healthy and diseased samples. While the causative agent(s) of contemporary WPX outbreaks remains elusive, our results suggest that Vibrio spp. may be part of a nonspecific heterotrophic bacterial bloom rather than acting as primary pathogens. This study highlights the need for highly resolved temporal sampling in situ to further elucidate the role of Vibrio during WPX onset and progression. IMPORTANCE Coral diseases are increasing worldwide and are now considered a major contributor to coral reef decline. In particular, the Caribbean has been noted as a coral disease hot spot, owing to the dramatic loss of framework-building acroporid corals due to tissue loss diseases. The pathogenesis of contemporary white pox disease (WPX) outbreaks in Acropora palmata remains poorly understood. This study investigates the association of Vibrio bacteria with WPX.
, Gustavo M. de Billerbeck, Jin-Jing Zhang, Frank Rosenzweig,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01553-17

Abstract:
Homology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). Yeast AAD genes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes, AAD4 and AAD14 , encode functional enzymes that reduce aliphatic and aryl-aldehydes concomitant with the oxidation of cofactor NADPH, and that Aad4p and Aad14p exhibit different substrate preference patterns. Other yeast AAD genes are undergoing pseudogenization. The 5′ sequence of AAD15 has been deleted from the genome. Repair of an AAD3 missense mutation at the catalytically essential Tyr 73 residue did not result in a functional enzyme. However, ancestral-state reconstruction by fusing Aad6 with Aad16 and by N-terminal repair of Aad10 restores NADPH-dependent aryl-alcohol dehydrogenase activities. Phylogenetic analysis indicates that AAD genes are narrowly distributed in wood-saprophyte fungi and in yeast that occupy lignocellulosic niches. Because yeast AAD genes exhibit activity on veratraldehyde, cinnamaldehyde, and vanillin, they could serve to detoxify aryl-aldehydes released during lignin degradation. However, none of these compounds induce yeast AAD gene expression, and Aad activities do not relieve aryl-aldehyde growth inhibition. Our data suggest an ancestral role for AAD genes in lignin degradation that is degenerating as a result of yeast's domestication and use in brewing, baking, and other industrial applications. IMPORTANCE Functional characterization of hypothetical genes remains one of the chief tasks of the postgenomic era. Although the first Saccharomyces cerevisiae genome sequence was published over 20 years ago, 22% of its estimated 6,603 open reading frames (ORFs) remain unverified. One outstanding example of this category of genes is the enigmatic seven-member AAD family. Here, we demonstrate that proteins encoded by two members of this family exhibit aliphatic and aryl-aldehyde reductase activity, and further that such activity can be recovered from pseudogenized AAD genes via ancestral-state reconstruction. The phylogeny of yeast AAD genes suggests that these proteins may have played an important ancestral role in detoxifying aromatic aldehydes in ligninolytic fungi. However, in yeast adapted to niches rich in sugars, AAD genes become subject to mutational erosion. Our findings shed new light on the selective pressures and molecular mechanisms by which genes undergo pseudogenization.
Yahong Wei, Jing Fu, Jianying Wu, Xinmiao Jia, Yunheng Zhou, Cuidan Li, Mengxing Dong, Shanshan Wang, ,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01898-17

Abstract:
Polyvinyl alcohol (PVA) is used widely in industry, and associated environmental pollution is a serious problem. Herein, we report a novel, efficient PVA degrader, Stenotrophomonas rhizophila QL-P4, isolated from fallen leaves from a virgin forest in the Qinling Mountains. The complete genome was obtained using single-molecule real-time (SMRT) technology and corrected using Illumina sequencing. Bioinformatics analysis revealed eight PVA/vinyl alcohol oligomer (OVA)-degrading genes. Of these, seven genes were predicted to be involved in the classic intracellular PVA/OVA degradation pathway, and one (BAY15_3292) was identified as a novel PVA oxidase. Five PVA/OVA-degrading enzymes were purified and characterized. One of these, BAY15_1712, a PVA dehydrogenase (PVADH), displayed high catalytic efficiency toward PVA and OVA substrate. All reported PVADHs only have PVA-degrading ability. Most importantly, we discovered a novel PVA oxidase (BAY15_3292) that exhibited higher PVA-degrading efficiency than the reported PVADHs. Further investigation indicated that BAY15_3292 plays a crucial role in PVA degradation in S. rhizophila QL-P4. Knocking out BAY15_3292 resulted in a significant decline in PVA-degrading activity in S. rhizophila QL-P4. Interestingly, we found that BAY15_3292 possesses exocrine activity, which distinguishes it from classic PVADHs. Transparent circle experiments further proved that BAY15_3292 greatly affects extracellular PVA degradation in S. rhizophila QL-P4. The exocrine characteristics of BAY15_3292 facilitate its potential application to PVA bioremediation. In addition, we report three new efficient secondary alcohol dehydrogenases (SADHs) with OVA-degrading ability in S. rhizophila QL-P4; in contrast, only one OVA-degrading SADH was reported previously. IMPORTANCE With the widespread application of PVA in industry, PVA-related environmental pollution is an increasingly serious issue. Because PVA is difficult to degrade, it accumulates in aquatic environments and causes chronic toxicity to aquatic organisms. Biodegradation of PVA, as an economical and environment-friendly method, has attracted much interest. To date, effective and applicable PVA-degrading bacteria/enzymes have not been reported. Herein, we report a new efficient PVA degrader ( S. rhizophila QL-P4) that has five PVA/OVA-degrading enzymes with high catalytic efficiency, among which BAY15_1712 is the only reported PVADH with both PVA- and OVA-degrading abilities. Importantly, we discovered a novel PVA oxidase (BAY15_3292) that is not only more efficient than other reported PVA-degrading PVADHs but also has exocrine activity. Overall, our findings provide new insight into PVA-degrading pathways in microorganisms and suggest S. rhizophila QL-P4 and its enzymes have the potential for application to PVA bioremediation to reduce or eliminate PVA-related environmental pollution.
Gautam Kamini, Dasvit Shetty, Vikas D. Trivedi, Madhushri Varunjikar,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.02115-17

Abstract:
Pseudomonas sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of mcbA , encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm of Escherichia coli . The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis of E. coli expressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparent K m and Vmax in the range of 98 to 111 μM and 69 to 73 μmol · min −1 · mg −1 , respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm of Pseudomonas spp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol. IMPORTANCE Proteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm of Pseudomonas spp. Predicted transmembrane domain and signal peptide sequences of Pseudomonas were found to be functional in Escherichia coli and to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.
C. Hartard, M. Leclerc, , A. Maul, J. Loutreul, S. Banas, N. Boudaud,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01866-17

Abstract:
Norovirus (NoV) is the leading cause of gastroenteritis outbreaks linked to oyster consumption. In this study, we investigated the potential of F-specific RNA bacteriophages (FRNAPH) as indicators of viral contamination in oysters by focusing especially on FRNAPH subgroup II (FRNAPH-II). These viral indicators have been neglected because their behavior is sometimes different from that of NoV in shellfish, especially during the depuration processes usually performed before marketing. However, a significant bias needs to be taken into account. This bias is that, in the absence of routine culture methods, NoV is targeted by genome detection, while the presence of FRNAPH is usually investigated by isolation of infectious particles. In this study, by targeting both viruses using genome detection, a significant correlation between the presence of FRNAPH-II and that of NoV in shellfish collected from various European harvesting areas impacted by fecal pollution was observed. Moreover, during their depuration, while the long period of persistence of NoV was confirmed, a similar or even longer period of persistence of the FRNAPH-II genome, which was over 30 days, was observed. Such a striking genome persistence calls into question the relevance of molecular methods for assessing viral hazards. Targeting the same virus (i.e., FRNAPH-II) by culture and genome detection in specimens from harvesting areas as well as during depuration, we concluded that the presence of genomes in shellfish does not provide any information on the presence of the corresponding infectious particles. In view of these results, infectious FRNAPH detection should be reconsidered as a valuable indicator in oysters, and its potential for use in assessing viral hazard needs to be investigated. IMPORTANCE This work brings new data about the behavior of viruses in shellfish, as well as about the relevance of molecular methods for their detection and evaluation of the viral hazard. First, a strong correlation between the presence of F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and that of norovirus (NoV) in shellfish impacted by fecal contamination has been observed when both viruses are detected using molecular approaches. Second, when reverse transcription-PCR and culture are used to detect FRNAPH-II in shellfish, it appears that the genomes of the viruses present a longer period of persistence than infectious virus, and thus, virus genome detection fails to give information about the concomitant presence of infectious viruses. Finally, this study shows that FRNAPH persist at least as long as NoV does. These data are major arguments to reconsider the potential of FRNAPH as indicators of shellfish viral quality.
Qiangde Duan, Jiachen Huang, Nan Xiao, Hyesuk Seo,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01737-17

Abstract:
Heat-stable toxin (STa)-producing enterotoxigenic Escherichia coli (ETEC) strains are a top cause of moderate-to-severe diarrhea in children from developing countries and a common cause of travelers' diarrhea. Recent progress in using STa toxoids and toxoid fusions to induce neutralizing anti-STa antibodies has accelerated ETEC vaccine development. However, concern remains regarding whether the derived anti-STa antibodies cross-react with STa-like guanylin and uroguanylin, two guanylate cyclase C (GC-C) ligands regulating fluid and electrolyte transportation in human intestinal and renal epithelial cells. To further divert STa from guanylin and uroguanylin structurally and antigenically and to eliminate anti-STa antibody cross-reactivity with guanylin and uroguanylin, we mutated STa at the 9th (leucine), 12th (asparagine), and 14th (alanine) residues for the double and triple mutants STa L9A/N12S , STa L9A/A14H , STa N12S/A14T , and STa L9A/N12S/A14H . We then fused each STa mutant (three copies) to a monomeric heat-labile toxin (LT) mutant (mnLT R192G/L211A ) for the toxoid fusions 3×STa L9A/N12S -mnLT R192G/L211A , 3×STa L9A/A14H -mnLT R192G/L211A , 3×STa N12S/A14T -mnLT R192G/L211A , and 3×STa L9A/N12S/A14H -mnLT R192G/L211A ; examined each fusion for anti-STa immunogenicity; and assessed the derived antibodies for in vitro neutralization activity against STa toxicity and for cross-reactivity with guanylin and uroguanylin. Mice subcutaneously immunized with each fusion protein developed anti-STa antibodies, and the antibodies derived from 3×STa N12S -mnLT R192G/L211A , 3×STa L9A/N12S -mnLT R192G/L211A , or 3×STa N12S/A14T -mnLT R192G/L211A prevented STa from the stimulation of intracellular cGMP in T-84 cells. Competitive enzyme-linked immunosorbent assays (ELISAs) showed that guanylin and uroguanylin hardly blocked the binding of anti-STa antibodies to the coated STa-ovalbumin conjugate. These results indicated that antibodies derived from 3×STa N12S -mnLT R192G/L211A , 3×STa L9A/N12S -mnLT R192G/L211A , or 3×STa N12S/A14T -mnLT R192G/L211A neutralized STa and had little cross-reactivity with guanylin and uroguanylin, suggesting that these toxoid fusions are suitable antigens for ETEC vaccines. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children's diarrhea and travelers' diarrhea. Currently, there is no licensed vaccine against ETEC diarrhea. One key challenge is to identify safe antigens to induce antibodies neutralizing the key STa without cross-reacting with guanylin and uroguanylin, two important ligands controlling homeostasis in human intestinal and renal epithelial cells. In this study, we generated nontoxic fusion antigens that induced antibodies that neutralize STa enterotoxicity in vitro and do not cross-react with guanylin or uroguanylin. These fusions have become the preferred antigens for the development of ETEC vaccines to potentially prevent the deaths of hundreds of thousands of young children and hundreds of millions of diarrheal cases each year.
Xingqun Cheng, , Nyssa Cullin, Xuedong Zhou, Xin Xu, Vrushali Joshi, Dipankar Koley, Justin Merritt,
Applied and Environmental Microbiology, Volume 84; https://doi.org/10.1128/aem.01697-17

Abstract:
Commensal Streptococcus sanguinis and Streptococcus gordonii are pioneer oral biofilm colonizers. Characteristic for both is the SpxB-dependent production of H 2 O 2 , which is crucial for inhibiting competing biofilm members, especially the cariogenic species Streptococcus mutans . H 2 O 2 production is strongly affected by environmental conditions, but few mechanisms are known. Dental plaque pH is one of the key parameters dictating dental plaque ecology and ultimately oral health status. Therefore, the objective of the current study was to characterize the effects of environmental pH on H 2 O 2 production by S. sanguinis and S. gordonii . S. sanguinis H 2 O 2 production was not found to be affected by moderate changes in environmental pH, whereas S. gordonii H 2 O 2 production declined markedly in response to lower pH. Further investigation into the pyruvate node, the central metabolic switch modulating H 2 O 2 or lactic acid production, revealed increased lactic acid levels for S. gordonii at pH 6. The bias for lactic acid production at pH 6 resulted in concomitant improvement in the survival of S. gordonii at low pH and seems to constitute part of the acid tolerance response of S. gordonii . Differential responses to pH similarly affect other oral streptococcal species, suggesting that the observed results are part of a larger phenomenon linking environmental pH, central metabolism, and the capacity to produce antagonistic amounts of H 2 O 2 . IMPORTANCE Oral biofilms are subject to frequent and dramatic changes in pH. S. sanguinis and S. gordonii can compete with caries- and periodontitis-associated pathogens by generating H 2 O 2 . Therefore, it is crucial to understand how S. sanguinis and S. gordonii adapt to low pH and maintain their competitiveness under acid stress. The present study provides evidence that certain oral bacteria respond to environmental pH changes by tuning their metabolic output in favor of lactic acid production, to increase their acid survival, while others maintain their H 2 O 2 production at a constant level. The differential control of H 2 O 2 production provides important insights into the role of environmental conditions for growth competition of the oral flora.
, Ricardo Torquato, Ricardo R. Brentani
Journal of Bacteriology, Volume 185, pp 4243-4247; https://doi.org/10.1128/jb.185.14.4243-4247.2003

Abstract:
In order to predict the binding regions within the complex formed by Toll-like receptor 5 (TLR-5) and flagellin, a complementary hydropathy between the two proteins was sought. A region common to the flagellins of Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa , and Listeria monocytogenes was shown to be hydropathically complementary to the 552-to-561 fragment of TLR-5, whose sequence is EILDISRNQL. The hydrophobicity profile of this region is shared with flagellins of 377 bacterial species out of a total of 723 publicly available sequences. A conformational analysis of the predicted binding site of TLR-5, whose structure is still unknown, was carried out with a methodology already applied to similar problems. To sample the conformations available to the peptide chain, a plot of the number of conformations per unit energy interval (density of states) versus energy was built. Following a theoretical argument, conformations belonging to maxima in this plot were selected. The most stable structure obtained in this search, an α-helical conformation, was shown to form the electrostatic interactions Glu552-Gln89, Asp555-Arg92, and Arg558-Glu93 with the predicted binding site of the flagellin of S . enterica serovar Typhimurium, formed by the 88-to-97 chain fragment (LQRVRELAVQ), which is likewise α helical.
, , Gianluca Fossati, Paolo Mascagni, , Steve P. Wood
Journal of Bacteriology, Volume 185, pp 4172-4185; https://doi.org/10.1128/jb.185.14.4172-4185.2003

Abstract:
The crystal structure of Mycobacterium tuberculosis chaperonin 10 (cpn10 Mt ) has been determined to a resolution of 2.8 Å. Two dome-shaped cpn10 Mt heptamers complex through loops at their bases to form a tetradecamer with 72 symmetry and a spherical cage-like structure. The hollow interior enclosed by the tetradecamer is lined with hydrophilic residues and has dimensions of 30 Å perpendicular to and 60 Å along the sevenfold axis. Tetradecameric cpn10 Mt has also been observed in solution by dynamic light scattering. Through its base loop sequence cpn10 Mt is known to be the agent in the bacterium responsible for bone resorption and for the contribution towards its strong T-cell immunogenicity. Superimposition of the cpn10 Mt sequences 26 to 32 and 66 to 72 and E. coli GroES 25 to 31 associated with bone resorption activity shows them to have similar conformations and structural features, suggesting that there may be a common receptor for the bone resorption sequences. The base loops of cpn10s in general also attach to the corresponding chaperonin 60 (cpn60) to enclose unfolded protein and to facilitate its correct folding in vivo. Electron density corresponding to a partially disordered protein subunit appears encapsulated within the interior dome cavity of each heptamer. This suggests that the binding of substrates to cpn10 is possible in the absence of cpn60.
Chia-Cheng Chou, Ting-Wan Lin, Chin-Yu Chen,
Journal of Bacteriology, Volume 185, pp 4066-4073; https://doi.org/10.1128/jb.185.14.4066-4073.2003

Abstract:
The crystal structure of a small, basic DNA binding protein, Sso10b2, from the thermoacidophilic archaeon Sulfolobus solfataricus was determined by the Zn multiwavelength anomalous diffraction method and refined to 1.85 Å resolution. The 89-amino-acid protein adopts a βαβαββ topology. The structure is similar to that of Sso10b1 (also called Alba) from the same organism. However, Sso10b2 contains an arginine-rich loop RDRRR motif, which may play an important role in nucleic acid binding. There are two independent Sso10b2 proteins in the asymmetric unit, and a plausible stable dimer could be deduced from the crystal structure. Topology comparison revealed that Sso10b2 is similar to several RNA-binding proteins, including IF3-C, YhhP, and DNase I. Models of the Sso10b2 dimer bound to either B-DNA or A-DNA have been constructed.
Kamaleldin E. Elagib, Mang Xiao, Isa M. Hussaini, Lorrie L. Delehanty, Lisa A. Palmer, Frederick K. Racke, Michael J. Birrer, Ganapath Shanmugasundaram, Michael A. McDevitt, Adam N. Goldfarb
Molecular and Cellular Biology, Volume 24, pp 7779-7794; https://doi.org/10.1128/mcb.24.17.7779-7794.2004

Abstract:
Although Jun upregulation and activation have been established as critical to oncogenesis, the relevant downstream pathways remain incompletely characterized. In this study, we found that c-Jun blocks erythroid differentiation in primary human hematopoietic progenitors and, correspondingly, that Jun factors block transcriptional activation by GATA-1, the central regulator of erythroid differentiation. Mutagenesis of c-Jun suggested that its repression of GATA-1 occurs through a transcriptional mechanism involving activation of downstream genes. We identified the hairy-enhancer-of-split-related factor HERP2 as a novel gene upregulated by c-Jun. HERP2 showed physical interaction with GATA-1 and repressed GATA-1 transcriptional activation. Furthermore, transduction of HERP2 into primary human hematopoietic progenitors inhibited erythroid differentiation. These results thus define a novel regulatory pathway linking the transcription factors c-Jun, HERP2, and GATA-1. Furthermore, these results establish a connection between the Notch signaling pathway, of which the HERP factors are a critical component, and the GATA family, which participates in programming of cellular differentiation.
William F. Schwindinger, Kathryn E. Giger, Kelly S. Betz, Anna M. Stauffer, Elaine M. Sunderlin, Laura J. Sim-Selley, Dana E. Selley, ,
Molecular and Cellular Biology, Volume 24, pp 7758-7768; https://doi.org/10.1128/mcb.24.17.7758-7768.2004

Abstract:
Emerging evidence suggests that the γ subunit composition of an individual G protein contributes to the specificity of the hundreds of known receptor signaling pathways. Among the twelve γ subtypes, γ 3 is abundantly and widely expressed in the brain. To identify specific functions and associations for γ 3 , a gene-targeting approach was used to produce mice lacking the Gng3 gene ( Gng3−/− ). Confirming the efficacy and specificity of gene targeting, Gng3−/− mice show no detectable expression of the Gng3 gene, but expression of the divergently transcribed Bscl2 gene is not affected. Suggesting unique roles for γ 3 in the brain, Gng3−/− mice display increased susceptibility to seizures, reduced body weights, and decreased adiposity compared to their wild-type littermates. Predicting possible associations for γ 3 , these phenotypic changes are associated with significant reductions in β 2 and α i3 subunit levels in certain regions of the brain. The finding that the Gng3−/− mice and the previously reported Gng7−/− mice display distinct phenotypes and different αβγ subunit associations supports the notion that even closely related γ subtypes, such as γ 3 and γ 7 , perform unique functions in the context of the organism.
Aiwen Jin, , Kevin O'Keefe,
Molecular and Cellular Biology, Volume 24, pp 7669-7680; https://doi.org/10.1128/mcb.24.17.7669-7680.2004

Abstract:
The importance of coordinating cell growth with proliferation has been recognized for a long time. The molecular basis of this relationship, however, is poorly understood. Here we show that the ribosomal protein L23 interacts with HDM2. The interaction involves the central acidic domain of HDM2 and an N-terminal domain of L23. L23 and L11, another HDM2-interacting ribosomal protein, can simultaneously yet distinctly interact with HDM2 together to form a ternary complex. We show that, when overexpressed, L23 inhibits HDM2-induced p53 polyubiquitination and degradation and causes a p53-dependent cell cycle arrest. On the other hand, knocking down L23 causes nucleolar stress and triggers translocation of B23 from the nucleolus to the nucleoplasm, leading to stabilization and activation of p53. Our data suggest that cells may maintain a steady-state level of L23 during normal growth; alternating the levels of L23 in response to changing growth conditions could impinge on the HDM2-p53 pathway by interrupting the integrity of the nucleolus.
Mu-Shui Dai, Shelya X. Zeng, Yetao Jin, Xiao-Xin Sun, Larry David,
Molecular and Cellular Biology, Volume 24, pp 7654-7668; https://doi.org/10.1128/mcb.24.17.7654-7668.2004

Abstract:
The p53-MDM2 feedback loop is vital for cell growth control and is subjected to multiple regulations in response to various stress signals. Here we report another regulator of this loop. Using an immunoaffinity method, we purified an MDM2-associated protein complex that contains the ribosomal protein L23. L23 interacted with MDM2, forming a complex independent of the 80S ribosome and polysome. The interaction of L23 with MDM2 was enhanced by treatment with actinomycin D but not by gamma-irradiation, leading to p53 activation. This activation was inhibited by small interfering RNA against L23. Ectopic expression of L23 reduced MDM2-mediated p53 ubiquitination and also induced p53 activity and G 1 arrest in p53-proficient U2OS cells but not in p53-deficient Saos-2 cells. These results reveal that L23 is another regulator of the p53-MDM2 feedback regulation.
Weicheng Wu, Raymond D. Mosteller,
Molecular and Cellular Biology, Volume 24, pp 7359-69; https://doi.org/10.1128/mcb.24.17.7359-7369.2004

Abstract:
Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria. Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs). LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1. Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA). Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment. Inhibition of SPK by the pharmacological inhibitor N , N -dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation. The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-κB reporters in RAW 264.7 cells. Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis. These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria.
, James Versalovic
Microbiology Spectrum, Volume 5; https://doi.org/10.1128/microbiolspec.bad-0012-2016

Abstract:
Commensal and beneficial microbes secrete myriad products which target the mammalian host and other microbes. These secreted substances aid in bacterial niche development, and select compounds beneficially modulate the host and promote health. Microbes produce unique compounds which can serve as signaling factors to the host, such as biogenic amine neuromodulators, or quorum-sensing molecules to facilitate inter-bacterial communication. Bacterial metabolites can also participate in functional enhancement of host metabolic capabilities, immunoregulation, and improvement of intestinal barrier function. Secreted products such as lactic acid, hydrogen peroxide, bacteriocins, and bacteriocin-like substances can also target the microbiome. Microbes differ greatly in their metabolic potential and subsequent host effects. As a result, knowledge about microbial metabolites will facilitate selection of next-generation probiotics and therapeutic compounds derived from the mammalian microbiome. In this article we describe prominent examples of microbial metabolites and their effects on microbial communities and the mammalian host.
, Bärbel Kiesel, Jakob Zopfi, ,
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.00914-17

Abstract:
Alteromonas virus vB_AspP-H4/4 is a member of the Podoviridae family and was isolated from North Sea water in the 1970s. The complete double-stranded DNA genome has 47,631 bp with 49 predicted genes.
, Dung Tuan Nguyen, Penghao Wang, Mitchell Andrews
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.00958-17

Abstract:
We report here the complete genome sequence of Mesorhizobium sophorae ICMP 19535 T . This strain was isolated from Sophora microphylla root nodules and can nodulate and fix nitrogen with this host and also with Sophora prostrata , Sophora longicarinata , and Clianthus puniceus . The genome consists of 8.05 Mb.
, Anna Maria Gebauer, Martin Hofrichter, , Harald Kellner
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01076-17

Abstract:
We report here the draft genome of Kretzschmaria ( Ustulina ) deusta , an ascomycetous fungus that colonizes and substantially degrades hardwood and can infest living broad-leaved trees. The genome was assembled into 858 contigs, with a total size of 46.5 Mb, and 11,074 protein-coding genes were predicted.
, , John Cushman, Jeremy Schmutz, Duc Tran, Leyla T. Hathwaik, , Jerry Jenkins, , Simon Prochnik, et al.
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01105-17

Abstract:
The halotolerant alga Dunaliella salina is a model for stress tolerance and is used commercially for production of beta-carotene (=pro-vitamin A). The presented draft genome of the genuine strain CCAP19/18 will allow investigations into metabolic processes involved in regulation of stress responses, including carotenogenesis and adaptations to life in high-salinity environments.
, Christina Tryfonos, Aristomenis Tourvas, Dora Floridou, Niki I. Paphitou, Christina Christodoulou
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01110-17

Abstract:
We report here the complete genome sequence of a West Nile virus (WNV) strain from the first laboratory-confirmed human case of neuroinvasive WNV infection in Cyprus. Phylogenetic analysis showed that this WNV strain grouped clearly into genetic lineage 1, clade 1a, cluster 2.
, Annelies Michem, Martine Peeters, Francis Vercammen
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01117-17

Abstract:
There are four lineages of primate T-cell lymphocytic viruses (human T-cell lymphocytic virus [HTLV]/simian T-cell lymphocytic virus [STLV]), which are further divided into subtypes. To date, there is only one full-length HTLV-1 subtype b genome available. Here, we report the genome of a new STLV-1 subtype b from a 43-year-old male gorilla with T-cell lymphoma.
Gaby Carl, Claudia Jäckel, , Stefan Hertwig, , Burkhard Malorny, Jörg Rau, Annemarie Käsbohrer,
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01118-17

Abstract:
We describe here the genome sequence of the novel temperate Klebsiella pneumoniae phage KPP5665-2 isolated from a Klebsiella pneumoniae strain recovered from milk in Germany in 2016. The phage exhibited a narrow host range and a siphoviridal morphology. KPP5665-2-related prophage sequences were detected in whole-genome sequencing (WGS) data of various Klebsiella species isolates.
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01134-17

Abstract:
Wolbachia spp. are endosymbiotic bacteria that infect around 50% of arthropods and cause a broad range of effects, including manipulating host reproduction. Here, we present the annotated draft genome assembly of Wolbachia strain wAus, which infects Plutella australiana , a cryptic ally of the major Brassica pest Plutella xylostella (diamondback moth).
, , M. Al Amin, Huzzat Ullah, , Samina Momtaz, Munawar Sultana,
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01135-17

Abstract:
The complete genome sequence of foot-and-mouth disease virus (FMDV) serotype Asia1 isolated from Bangladesh is reported here. Genome analysis revealed amino acid substitutions in the VP1 antigenic region and deletions in both the 5′ and 3′ untranslated regions (UTRs) compared to the genome of the existing vaccine strain (GenBank accession no. AY304994).
, , Yuki Furuse, Robert H. Gilman, Xiaofang Liu, Sonia Apaza, Susan Espetia, Vitaliano Cama, Hitoshi Oshitani,
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01137-17

Abstract:
We report here two complete coding genome sequences of novel genotype GII.8 sapovirus strains identified in diarrhea samples collected from two Peruvian children. The complete coding genome sequences of both GII.8 variants were determined using the Sanger sequencing method.
, Vania C. Liuzzi, , , , Antonio F. Logrieco,
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01141-17

Abstract:
Pseudomonas fluorescens is a genetically and phenotypically heterogeneous species that is often reported as a spoiler of fresh foods, but it has recently been implicated in clinical infection. In this study, we sequenced the genome of P. fluorescens strain ITEM 17298, isolated from mozzarella cheese and able to cause several alterations under cold storage.
, Munesh Kumari, Rakesh Kochhar, Devinder Kumar Dhawan, , Shanmugam Mayilraj
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01158-17

Abstract:
We report here the 3.8-Mb genome sequence of Kocuria polaris strain CD08_4, an isolate from the duodenal mucosa of a celiac disease patient. The genome consists of specific virulence determinant genes, antibiotic resistance genes, genes for coping with oxidative stress, and genes responsible for iron acquisition and metabolism, suggestive of its pathogenic attributes.
, Jinglin Chang, Xin He, Qinlian Hou, Wei Long
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01163-17

Abstract:
Bacillus subtilis CGMCC 12426 is an efficient producer of poly-γ-glutamate with regular stereochemistry. Here, the complete genome sequence of B. subtilis CGMCC 12426 is presented, which may facilitate the design of rational strategies for further strain improvements with industrial potential.
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01167-17

Abstract:
The complete genome sequence of Eubacterium hallii strain L2-7 is reported here. This intestinal strain produces butyrate from glucose as well as lactate when acetate is provided in the growth medium. In addition, strain L2-7 has been shown to improve insulin sensitivity in db/db mice, indicating its application potential.
Kevin Egan, , , , R. Paul Ross, ,
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01172-17

Abstract:
This paper reports the full genome sequence of the antimicrobial-producing bacterium Geobacillus stearothermophilus DSM 458, isolated in a sugar beet factory in Austria. In silico analysis reveals the presence of a number of novel bacteriocin biosynthetic genes.
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01174-17

Abstract:
Epizootic hemorrhagic disease virus (EHDV) serotypes 1 and 2 were isolated from Florida white-tailed deer in 2015 and 2016, respectively, and their genomes were completely sequenced. To our knowledge, these are the first full genome sequences for EHDV-1 and -2 from Florida.
Kristen A. Butela, Susan M. R. Gurney, , Janine M. LeBlanc-Straceski, Anastasia M. Zimmerman, Stephanie B. Conant, Nikki E. Freed, Olin K. Silander, , Charlotte A. Berkes, et al.
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01182-17

Abstract:
Seven mycobacteriophages from distinct geographical locations were isolated, using Mycobacterium smegmatis mc 2 155 as the host, and then purified and sequenced. All of the genomes are related to cluster A mycobacteriophages, BobSwaget and Lokk in subcluster A2; Fred313, KADY, Stagni, and StepMih in subcluster A3; and MyraDee in subcluster A18, the first phage to be assigned to that subcluster.
Zaratulnur Mohd Bahari, Zaharah Ibrahim, Jafariah Jaafar,
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01183-17

Abstract:
Microbacterium sp. strain SZ1 isolated from gold ores of a Malaysia gold mine was found to be highly resistant to arsenic. Here, we report the draft genome sequence of SZ1, which may provide further insights into understanding its arsenic resistance mechanism. In this draft genome, a complete set of ars operons and two additional scattered ars genes were encoded.
, , Paul Zappile, , , Adriana Heguy, Victor Tetz
Genome Announcements, Volume 5; https://doi.org/10.1128/genomea.01184-17

Abstract:
We report here an update to the draft genome sequence of Kluyvera intestini sp. nov. strain GT-16, generated using MinION long-read sequencing technology. The complete genome sequence of the human-derived strain GT-16 measured 5,768,848 bp. An improved high-quality complete genome sequence provides insights into the mobility potential of resistance genes in this species.
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