Legionella pneumophila, the gram-negative agent of Legionnaires' disease, possesses type IV pili and a type II protein secretion (Lsp) system, both of which are dependent upon the PilD prepilin peptidase. By analyzing multiple pilD mutants and various types of Lsp mutants as well as performing trans-complementation of these mutants, we have confirmed that PilD and type II secretion genes are required for L. pneumophila infection of both amoebae and human macrophages. Based upon a complete analysis of lspDE, lspF, and lspG mutants, we found that the type II system controls the secretion of protease, RNase, lipase, phospholipase A, phospholipase C, lysophospholipase A, and tartrate-sensitive and tartrate-resistant acid phosphatase activities and influences the appearance of colonies. Examination of the developing L. pneumophila genome database indicated that the organism has two other loci (lspC and lspLM) that are predicted to promote secretion and thus a set of genes that is comparable to the type II secretion genes in other gram-negative bacteria. In contrast to lsp mutants, L. pneumophila pilus mutants lacking either the PilQ secretin, the PspA pseudopilin, or pilin were not defective for colonial growth, secreted activities, or intracellular replication. L. pneumophila dot/icm mutants were also not impaired for type II-dependent exoenzymes. Upon intratracheal inoculation into A/J mice, lspDE, lspF, and pilD mutants, but not pilus mutants, exhibited a reduced ability to grow in the lung, as measured by competition assays. The lspF mutant was also defective in an in vivo kinetic assay. Examination of infected mouse sera revealed that type II secreted proteins are expressed in vivo. Thus, the L. pneumophila Lsp system is a virulence factor and the only type II secretion system linked to intracellular infection.The gram-negative bacterium Legionella pneumophila is the agent of Legionnaires' disease, a pneumonia which especially affects immunocompromised individuals (28,89). An inhabitant of freshwater environments, L. pneumophila naturally replicates within protozoan hosts and in biofilms (27, 89). Following inhalation of contaminated aerosols, the bacterium reaches the human respiratory tract. Bacterial multiplication in alveolar macrophages is concomitant with cell death and damage to the lung tissue (89, 100).In gram-negative bacteria, the PilD prepilin peptidase is necessary for the cleavage and methylation of pilins and pseudopilins that assemble into type IV pili (Tfp) (55,68,69,87). In addition, PilD processes other pseudopilins that are necessary for the biogenesis of a functional type II protein secretion system (8,13,55,71,86). Accordingly, our previous mutational analysis determined that pilD is required for L. pneumophila piliation and protein secretion (4, 52). In L. pneumophila, Tfp promote attachment to host cells and are involved in competence for DNA transformation (84, 85). The L. pneumophila proteins believed to be secreted via the type II system include a zinc metalloprotease, acid phosphatase...
The complete genome of the ammonia-oxidizing bacterium Nitrosospira multiformis (ATCC 25196 T ) consists of a circular chromosome and three small plasmids totaling 3,234,309 bp and encoding 2,827 putative proteins. Of the 2,827 putative proteins, 2,026 proteins have predicted functions and 801 are without conserved functional domains, yet 747 of these have similarity to other predicted proteins in databases. Gene homologs from Nitrosomonas europaea and Nitrosomonas eutropha were the best match for 42% of the predicted genes in N. multiformis. The N. multiformis genome contains three nearly identical copies of amo and hao gene clusters as large repeats. The features of N. multiformis that distinguish it from N. europaea include the presence of gene clusters encoding urease and hydrogenase, a ribulose-bisphosphate carboxylase/oxygenase-encoding operon of distinctive structure and phylogeny, and a relatively small complement of genes related to Fe acquisition. Systems for synthesis of a pyoverdine-like siderophore and for acyl-homoserine lactone were unique to N. multiformis among the sequenced genomes of ammonia-oxidizing bacteria. Gene clusters encoding proteins associated with outer membrane and cell envelope functions, including transporters, porins, exopolysaccharide synthesis, capsule formation, and protein sorting/export, were abundant. Numerous sensory transduction and response regulator gene systems directed toward sensing of the extracellular environment are described. Gene clusters for glycogen, polyphosphate, and cyanophycin storage and utilization were identified, providing mechanisms for meeting energy requirements under substrate-limited conditions. The genome of N. multiformis encodes the core pathways for chemolithoautotrophy along with adaptations for surface growth and survival in soil environments.Nitrification is a key process in the nitrogen cycle of terrestrial, wastewater, and marine systems. The first step in the aerobic process is the oxidation of ammonia, mediated by ammonia-oxidizing bacteria (AOB) or ammonia-oxidizing archaea. Because we are particularly interested in the genetic complement adaptive for ammonia-based chemolithotrophy in the soil environment, we completed the genome sequence of the soil AOB Nitrosospira multiformis (ATCC 25196 T ). Obtaining the N. multiformis genome sequence offers a unique opportunity for comparison to the available genomes of other betaproteobacterial AOB (beta-AOB), Nitrosomonas europaea (16), and Nitrosomonas eutropha (63). The AOB isolated or detected by noncultural methods in aerobic surface soils all have been members of the Betaproteobacteria (order Nitrosomonadales, family Nitrosomonadaceae). Recent evidence suggests that Crenarchaeota may also contribute to ammonia oxidation in soils (43).The sequenced AOB, Nitrosospira multiformis ATCC 25196 T , was isolated from soil near Paramaribo, Surinam, by enrichment culturing, followed by serial dilution to extinction (71). Originally, this isolate was the type strain for Nitrosolobus multiformis, wit...
The alphaproteobacterium Nitrobacter winogradskyi (ATCC 25391) is a gram-negative facultative chemolithoautotroph capable of extracting energy from the oxidation of nitrite to nitrate. Sequencing and analysis of its genome revealed a single circular chromosome of 3,402,093 bp encoding 3,143 predicted proteins. There were extensive similarities to genes in two alphaproteobacteria, Bradyrhizobium japonicum USDA110 (1,300 genes) and Rhodopseudomonas palustris CGA009 CG (815 genes). Genes encoding pathways for known modes of chemolithotrophic and chemoorganotrophic growth were identified. Genes encoding multiple enzymes involved in anapleurotic reactions centered on C 2 to C 4 metabolism, including a glyoxylate bypass, were annotated. The inability of N. winogradskyi to grow on C 6 molecules is consistent with the genome sequence, which lacks genes for complete Embden-Meyerhof and Entner-Doudoroff pathways, and active uptake of sugars. Two gene copies of the nitrite oxidoreductase, type I ribulose-1,5-bisphosphate carboxylase/oxygenase, cytochrome c oxidase, and gene homologs encoding an aerobictype carbon monoxide dehydrogenase were present. Similarity of nitrite oxidoreductases to respiratory nitrate reductases was confirmed. Approximately 10% of the N. winogradskyi genome codes for genes involved in transport and secretion, including the presence of transporters for various organic-nitrogen molecules. The N. winogradskyi genome provides new insight into the phylogenetic identity and physiological capabilities of nitrite-oxidizing bacteria. The genome will serve as a model to study the cellular and molecular processes that control nitrite oxidation and its interaction with other nitrogen-cycling processes.Nitrification, the microbiological process by which ammonia is converted to nitrate, is a major component of the global nitrogen cycle, plays a crucial role in transformation of fertilizer nitrogen in agricultural systems, and is a key component of nitrogen removal in wastewater treatment. Excess production of soluble nitrogen by nitrification results in the contamination of potable water and eutrophication of aquatic and terrestrial ecosystems, while the gaseous by-products of nitrification, nitric oxide and nitrous oxide, are two of the most potent greenhouse gases. As anthropogenic inputs of fixed nitrogen continue to expand to meet the demands of a growing global population, intimate knowledge of the nitrification process and the microorganisms that control this process will be necessary to address environmental nitrogen imbalances.Nitrobacter winogradskyi Nb-255 and other nitrite-oxidizing bacteria participate in nitrification by converting nitrite, the end product of ammonia oxidation, into nitrate according to the reaction NO 2 Ϫ ϩ H 2 O 3 NO 3 Ϫ ϩ 2H ϩ ϩ 2e Ϫ . Nitrite functions as an electron donor for the reduction of NAD via reverse electron flow and the generation of ATP by oxidative phosphorylation (36).As a facultative chemolithoautotroph, N. winogradskyi gains energy from nitrite oxidation, and fix...
The alphaproteobacterium Nitrobacter hamburgensis X14 is a gram-negative facultative chemolithoautotroph that conserves energy from the oxidation of nitrite to nitrate. Sequencing and analysis of the Nitrobacter hamburgensis X14 genome revealed four replicons comprised of one chromosome (4.4 Mbp) and three plasmids (294, 188, and 121 kbp). Over 20% of the genome is composed of pseudogenes and paralogs. Whole-genome comparisons were conducted between N. hamburgensis and the finished and draft genome sequences of Nitrobacter winogradskyi and Nitrobacter sp. strain Nb-311A, respectively. Most of the plasmid-borne genes were unique to N. hamburgensis and encode a variety of functions (central metabolism, energy conservation, conjugation, and heavy metal resistance), yet ϳ21 kb of a ϳ28-kb "autotrophic" island on the largest plasmid was conserved in the chromosomes of Nitrobacter winogradskyi Nb-255 and Nitrobacter sp. strain Nb-311A. The N. hamburgensis chromosome also harbors many unique genes, including those for heme-copper oxidases, cytochrome b 561 , and putative pathways for the catabolism of aromatic, organic, and one-carbon compounds, which help verify and extend its mixotrophic potential. A Nitrobacter "subcore" genome was also constructed by removing homologs found in strains of the closest evolutionary relatives, Bradyrhizobium japonicum and Rhodopseudomonas palustris. Among the Nitrobacter subcore inventory (116 genes), copies of genes or gene clusters for nitrite oxidoreductase (NXR), cytochromes associated with a dissimilatory nitrite reductase (NirK), PII-like regulators, and polysaccharide formation were identified. Many of the subcore genes have diverged significantly from, or have origins outside, the alphaproteobacterial lineage and may indicate some of the unique genetic requirements for nitrite oxidation in Nitrobacter.Nitrification is a two-step process by which ammonia is converted to nitrate via nitrite. Nitrification plays a key role in transformation of fertilizer nitrogen in agricultural systems and is a key component of nitrogen removal in wastewater treatment. Production of soluble inorganic nitrogen by nitrification can lead to the contamination and eutrophication of terrestrial and aquatic ecosystems, while the gaseous products of nitrifier denitrification, nitric oxide (NO) and nitrous oxide (N 2 O), destroy stratospheric ozone and greatly contribute to global warming (26, 54, 61). Nitrite-oxidizing bacteria (NOB) participate in the process of nitrification by converting nitrite (NO 2 Ϫ ), the end product of ammonia oxidation, into nitrate (NO 3 Ϫ ) according to the following reaction; NO 2Nitrite also functions as an electron donor for the reduction of NAD ϩ via reverse electron flow as well as for the generation of ATP by oxidative phosphorylation (17).Although several phylogenetically distinct genera (Nitrospira, Nitrobacter, Nitrococcus, and Nitrospina) carry out NO 2 Ϫ oxidation, most of what is known about the physiology and biochemistry of NOB has been derived from studies of t...
Legionella pneumophila type II secretion mutants showed reduced survival in both tap water at 4 to 17°C and aquatic amoebae at 22 to 25°C. Wild-type supernatants stimulated the growth of these mutants, indicating that secreted factors promote low-temperature survival. There was a correlation between low-temperature survival and secretion function when 12 additional Legionella species were examined.Legionella pneumophila is widespread in natural and manmade water systems (8,21,29,39,41,47,53,64). In these habitats, L. pneumophila exists planktonically, within protozoa, and in biofilms (14,35,36,41,45). The ubiquity of L. pneumophila is also a result of the organism's ability to survive at many temperatures, including ones as low as 4°C (21,29,62,64). L. pneumophila is an important pathogen of humans, with the inhalation of contaminated water droplets originating from aerosol-generating devices resulting in Legionnaires' disease (16). Given the manner in which infection occurs, it is important to better understand how legionellae survive in water, in protozoa, and at low temperatures. Recently, we found that L. pneumophila type II protein secretion is critical for growth in rich broth or agar at 12 to 25°C but not in medium at 30 to 37°C (56). Operative in many gram-negatives (9), type II secretion is a multistep process in which proteins are translocated across the inner membrane in a Sec-or Tat-dependent manner, recognized in the periplasm, and then delivered to the T2S apparatus, whereupon a pilus-like structure "pushes" proteins through a dedicated outer membrane pore or secretin (28).To investigate the connection between type II secretion and low-temperature survival under conditions that more closely mimic natural habitats, we compared wild-type serogroup 1 strain 130b (Table 1) and its type II secretion mutants for persistence in tap water incubated at 37°C, 25°C, and 17°C. We used three mutants: NU258, containing a mutation in the genes encoding the type II outer membrane secretin (lspD) and the inner membrane ATPase (lspE); NU275, containing a mutation in the gene for the inner membrane platform protein (lspF); and NU272 mutated in the gene encoding the pseudopilin peptidase (pilD) (51). Tap water was obtained from laboratory sinks and filter sterilized. Following growth at 37°C in buffered yeast extract (BYE) broth to late log phase (56), wild types and mutants were inoculated into flasks containing 50 ml of the tap water, and then the cultures were incubated with shaking. As with other wild-type L. pneumophila (30,41,42,54,57), 130b persisted in low-temperature tap water for extended times (Fig. 1). Also similar to previous work (27), the recovery of CFU was maintained for a longer period at low temperatures below 37°C. But across the 17 to 37°C range, the secretion mutants behaved differently than their parent (Fig. 1). At 37°C, the mutants displayed a greater recoverability than 130b between days 7 and 20 (P Ͻ 0.05). In a similar vein, at 25°C, the mutants were recovered more than the wild type was be...
In developing rice () endosperm, mRNAs of the major storage proteins, glutelin and prolamine, are transported and anchored to distinct subdomains of the cortical endoplasmic reticulum. RNA binding protein RBP-P binds to both glutelin and prolamine mRNAs, suggesting a role in some aspect of their RNA metabolism. Here, we show that rice lines expressing mutant RBP-P mislocalize both glutelin and prolamine mRNAs. Different mutant RBP-P proteins exhibited varying degrees of reduced RNA binding and/or protein-protein interaction properties, which may account for the mislocalization of storage protein RNAs. In addition, partial loss of RBP-P function conferred a broad phenotypic variation ranging from dwarfism, chlorophyll deficiency, and sterility to late flowering and low spikelet fertility. Transcriptome analysis highlighted the essential role of RBP-P in regulating storage protein genes and several essential biological processes during grain development. Overall, our data demonstrate the significant roles of RBP-P in glutelin and prolamine mRNA localization and in the regulation of genes important for plant growth and development through its RNA binding activity and cooperative regulation with interacting proteins.
Microalgae are promising biocatalysts for applications in sustainable fuel, food, and chemical production. Here, we describe culture collection screening, down-selection, and development of a high-productivity, halophilic, thermotolerant microalga, Picochlorum renovo. This microalga displays a rapid growth rate and high diel biomass productivity (34 g m−2 day−1), with a composition well-suited for downstream processing. P. renovo exhibits broad salinity tolerance (growth at 107.5 g L−1 salinity) and thermotolerance (growth up to 40 °C), beneficial traits for outdoor cultivation. We report complete genome sequencing and analysis, and genetic tool development suitable for expression of transgenes inserted into the nuclear or chloroplast genomes. We further evaluate mechanisms of halotolerance via comparative transcriptomics, identifying novel genes differentially regulated in response to high salinity cultivation. These findings will enable basic science inquiries into control mechanisms governing Picochlorum biology and lay the foundation for development of a microalga with industrially relevant traits as a model photobiology platform.
The extent to which different sequence-based approaches describe environmental microbial communities in comparative studies is an important consideration when deriving inferences from ecological studies. The ability of a targeted metagenomic approach [small subunit (SSU) rRNA pyrosequencing] and shotgun metagenome approaches were compared to identify distinguishing features in dryland soil microbial communities from two different habitats: biological soil crusts (biocrusts) and creosote bush root zones. A parallel comparison was conducted to determine the ability of each approach to detect community differences potentially arising from a more subtle experimental treatment, long-term elevated atmospheric carbon dioxide. As expected, the biocrust datasets were clearly differentiated from root zone datasets using either of the sequencing approaches. However, the composition described by each approach was significantly different. The magnitude of comparative differences due to habitat or elevated CO2 treatment was larger with pyrosequenced SSU datasets or SSU reads recruited from shotgun metagenomes, than from SEED-classified shotgun metagenome reads. Finally, based on prior knowledge of the biocrust communities, the SSU-based datasets more accurately identified the dominant biocrust cyanobacteria sequences compared to the shotgun metagenome datasets.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.