The Complete Genome Sequence of Natrinema sp. J7-2, a Haloarchaeon Capable of Growth on Synthetic Media without Amino Acid Supplements

Natrinema sp. J7-2 is an extreme haloarchaeon capable of growing on synthetic media without amino acid supplements. Here we report the complete genome sequence of Natrinema sp. J7-2 which is composed of a 3,697,626-bp chromosome and a 95,989-bp plasmid pJ7-I. This is the first complete genome sequence of a member of the genus Natrinema. We demonstrate that Natrinema sp. J7-2 can use gluconate, glycerol, or acetate as the sole carbon source and that its genome encodes complete metabolic pathways for assimilating these substrates. The biosynthetic pathways for all 20 amino acids have been reconstructed, and we discuss a possible evolutionary relationship between the haloarchaeal arginine synthetic pathway and the bacterial lysine synthetic pathway. The genome harbors the genes for assimilation of ammonium and nitrite, but not nitrate, and has a denitrification pathway to reduce nitrite to N(2)O. Comparative genomic analysis suggests that most sequenced haloarchaea employ the TrkAH system, rather than the Kdp system, to actively uptake potassium. The genomic analysis also reveals that one of the three CRISPR loci in the Natrinema sp. J7-2 chromosome is located in an integrative genetic element and is probably propagated via horizontal gene transfer (HGT). Finally, our phylogenetic analysis of haloarchaeal genomes provides clues about evolutionary relationships of haloarchaea.     

Lateral gene transfer occurring in haloarchaea: an interpretative imitation study

Lateral gene transfer (LGT) plays an important role in the molecular evolution of haloarchaea. Polyethylene glycol-mediated LGT in haloarchaea has been demonstrated in the laboratory, yet few explanations have been put forward for the apparently common, natural occurrence of plentiful plasmids within haloarchaeal cells. In this study, LGT was induced in two genera of haloarchaea, Haloferax and Halorubrum, by modification of salt concentration of media-a factor that may vary naturally in native haloarchaeal habitat. Minimal growth salt concentrations (MGSCs) of four strains of haloarchaea from these two genera were established, and transformations using two circular double-stranded DNAs (dsDNAs), pSY1 and pWL102, were then produced in media at strain-appropriate MGSCs. The four strains of haloarchaea were transformed successfully by both kinds of dsDNAs with an efficiency of 10(2)-10(3) transformants per microgram dsDNA. The transformation under reduced salt concentration may be an imitation of natural LGT of dsDNA into haloarchaea when salinity in normally hypersaline environments is altered by sudden introduction of fresh water-for example, by rainfall, snow-melt, or flooding-providing a reasonable interpretation for haloarchaea being naturally richer in plasmids than any other known organisms.     

Wide distribution among halophilic archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type III synthases

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular carbon and energy storage polymers by various bacteria and a few haloarchaea. In this study, 28 strains belonging to 15 genera in the family Halobacteriaceae were investigated with respect to their ability to synthesize PHAs and the types of their PHA synthases. Fermentation results showed that 18 strains from 12 genera could synthesize polyhydroxybutyrate (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). For most of these haloarchaea, selected regions of the phaE and phaC genes encoding PHA synthases (type III) were cloned via PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and were sequenced. The PHA synthases were also examined by Western blotting using haloarchaeal Haloarcula marismortui PhaC (PhaC(Hm)) antisera. Phylogenetic analysis showed that the type III PHA synthases from species of the Halobacteriaceae and the Bacteria domain clustered separately. Comparison of their amino acid sequences revealed that haloarchaeal PHA synthases differed greatly in both molecular weight and certain conserved motifs. The longer C terminus of haloarchaeal PhaC was found to be indispensable for its enzymatic activity, and two additional amino acid residues (C143 and C190) of PhaC(Hm) were proved to be important for its in vivo function. Thus, we conclude that a novel subtype (IIIA) of type III PHA synthase with unique features that distinguish it from the bacterial subtype (IIIB) is widely distributed in haloarchaea and appears to be involved in PHA biosynthesis.     

Diversity,taxonomy, and evolution of archaeal viruses of the class Caudoviricetes

The archaeal tailed viruses (arTV), evolutionarily related to tailed double-stranded DNA (dsDNA) bacteriophages of the class Caudoviricetes, represent the most common isolates infecting halophilic archaea. Only a handful of these viruses have been genomically characterized, limiting our appreciation of their ecological impacts and evolution. Here, we present 37 new genomes of haloarchaeal tailed virus isolates, more than doubling the current number of sequenced arTVs. Analysis of all 63 available complete genomes of arTVs, which we propose to classify into 14 new families and 3 orders, suggests ancient divergence of archaeal and bacterial tailed viruses and points to an extensive sharing of genes involved in DNA metabolism and counterdefense mechanisms, illuminating common strategies of virus–host interactions with tailed bacteriophages. Coupling of the comparative genomics with the host range analysis on a broad panel of haloarchaeal species uncovered 4 distinct groups of viral tail fiber adhesins controlling the host range expansion. The survey of metagenomes using viral hallmark genes suggests that the global architecture of the arTV community is shaped through recurrent transfers between different biomes, including hypersaline, marine, and anoxic environments.

Comparative genomics and host range analysis reveals the remarkable diversity and evolution of tailed archaeal viruses of the order Caudoviricetes, which together with their bacterial relatives arguably represent the most abundant and widespread virus group on our planet.     

Evolution of osmoregulatory patterns and gill ion transport mechanisms in the decapod Crustacea: a review

Decapod crustaceans exhibit a wide range of osmoregulatory patterns and capabilities from marine osmoconformers to brackish and freshwater hyperregulators to terrestrial hyporegulators. The principal gill salt transport mechanisms proposed to underlie the ability of the better-known taxa to occupy these specific habitats are examined here. Traditional thinking suggests that a graduated series of successively stronger adaptive mechanisms may have driven the occupation of ever more dilute osmotic niches, culminating in the conquest of freshwater and dry land. However, when habitat and osmoregulatory parameters are analyzed quantitatively against the phylogenies of the taxa examined, as illustrated here using a palaemonid shrimp clade, their association becomes questionable and may hold true only in specific cases. We also propose a putative evolution for gill epithelial ion pump and transporter arrangement in a eubrachyuran crab clade whose lineages occupy distinct osmotic niches. By including the systematics of these selected groups, this review incorporates the notion of a protracted time scale, here termed ??phylophysiology??, into decapod osmoregulation, allowing the examination of putative physiological transformations and their underlying evolutionary processes. This approach assumes that species are temporally linked, a factor that can impart phylogenetic structuring, which must be considered in comparative studies. Future experimental models in decapod osmoregulatory physiology should contemplate the phylogenetic relationships among the taxa chosen to better allow comprehension of the transformations arising during their evolution.     

Novel insights into the diversity of catabolic metabolism from ten haloarchaeal genomes

Background

The extremely halophilic archaea are present worldwide in saline environments and have important biotechnological applications. Ten complete genomes of haloarchaea are now available, providing an opportunity for comparative analysis.

Methodology/Principal Findings

We report here the comparative analysis of five newly sequenced haloarchaeal genomes with five previously published ones. Whole genome trees based on protein sequences provide strong support for deep relationships between the ten organisms. Using a soft clustering approach, we identified 887 protein clusters present in all halophiles. Of these core clusters, 112 are not found in any other archaea and therefore constitute the haloarchaeal signature. Four of the halophiles were isolated from water, and four were isolated from soil or sediment. Although there are few habitat-specific clusters, the soil/sediment halophiles tend to have greater capacity for polysaccharide degradation, siderophore synthesis, and cell wall modification. Halorhabdus utahensis and Haloterrigena turkmenica encode over forty glycosyl hydrolases each, and may be capable of breaking down naturally occurring complex carbohydrates. H. utahensis is specialized for growth on carbohydrates and has few amino acid degradation pathways. It uses the non-oxidative pentose phosphate pathway instead of the oxidative pathway, giving it more flexibility in the metabolism of pentoses.

Conclusions

These new genomes expand our understanding of haloarchaeal catabolic pathways, providing a basis for further experimental analysis, especially with regard to carbohydrate metabolism. Halophilic glycosyl hydrolases for use in biofuel production are more likely to be found in halophiles isolated from soil or sediment.     

Ptk2 contributes to osmoadaptation in the filamentous fungus Neurospora crassa

Hyphal tip-growing organisms often rely upon an internal hydrostatic pressure (turgor) to drive localized expansion of the cell. Regulation of the turgor in response to osmotic shock is mediated primarily by an osmotic MAP kinase cascade which activates osmolyte synthesis and ion uptake to effect turgor recovery. We characterized a Neurospora crassa homolog (PTK2) of ser/thr kinase regulators of ion transport in yeast to determine its role in turgor regulation in a filamentous fungi. The ptk2 mutant is osmosensitive, and has lower turgor poise than wildtype. The cause appears to be lower activity of the plasma membrane H⁺-ATPase. Its role in osmoadaptation is unrelated to the activity of the osmotic MAP kinase cascade. Instead, it acts in an alternative pathway that, like the osmotic MAP kinase cascade, also involves ion transport mediated osmoadaptation.     

连云港台北和盐城三圩盐田土壤嗜盐菌多样性研究

盐田土壤嗜盐微生物对盐田生态系统的良性循环和盐的生产至关重要。本文对江苏连云港台北盐田土壤和盐城三圩盐田土壤的嗜盐细菌和古菌的多样性进行了研究, 结果表明两地盐土嗜盐细菌和古菌的分布具有相似性和独特性。采用培养法从两地盐土中共分离到17株嗜盐细菌, 其中Halomonas为两地盐土共有的嗜盐细菌, 而Halobacillus和Pontibacillus仅在三圩盐土中发现。通过非培养的16S rDNA 基因文库法从两地盐土中发现了13种嗜盐古菌, 台北盐土有Halobacterium 和 Haloplanus, 三圩盐土有Halobacterium, Natronobacterium, Halogeometricum 和 Haloarcula。10个嗜盐古菌的16S rDNA和GenBank已知序列的同源性为92%~97%, 可能为这些属中的新种。该研究为盐田环境嗜盐微生物资源的开发和利用奠定了基础。     

连云港台北和盐城三圩盐田土壤嗜盐菌多样性研究

盐田土壤嗜盐微生物对盐田生态系统的良性循环和盐的生产至关重要.本文对江苏连云港台北盐田土壤和盐城三圩盐田土壤的嗜盐细菌和古菌的多样性进行了研究,结果表明两地盐土嗜盐细菌和古菌的分布具有相似性和独特性.采用培养法从两地盐土中共分离到17株嗜盐细菌,其中Halomonas为两地盐土共有的嗜盐细菌,而Halobacillus和Pontibacillus仅在三圩盐土中发现.通过非培养的16S rDNA基因文库法从两地盐土中发现了13种嗜盐古菌,台北盐土有Halobacterium和Haloplanus,三圩盐土有Halobacterium, Natronobacterium, Halogeometricum和Haloarcula. 10个嗜盐古菌的16S rDNA和GenBank已知序列的同源性为92%～97%.可能为这些属中的新该研究为盐田环境嗜盐微生物资源的开发和利用奠定了基础.     

Osmotic stress response in <Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">glutamicum</Emphasis>: impact of channel- and transporter-mediated potassium accumulation

Potassium accumulation is an essential aspect of bacterial response to diverse stress situations; consequently its uptake plays a pivotal role. Here, we show that the Gram-positive soil bacterium Corynebacterium glutamicum which is employed for the large-scale industrial production of amino acids requires potassium under conditions of ionic and non-ionic osmotic stress. Besides the accumulation of high concentrations of potassium contributing significantly to the osmotic potential of the cytoplasm, we demonstrate that glutamate is not the counter ion for potassium under these conditions. Interestingly, potassium is required for the activation of osmotic stress-dependent expression of the genes betP and proP. The Kup-type potassium transport system which is present in C. glutamicum in addition to the potassium channel CglK does not contribute to potassium uptake at conditions of hyperosmotic stress. Furthermore, we established a secondary carrier of the KtrAB type from C. jeikeium in C. glutamicum thus providing an experimental comparison of channel- and carrier-mediated potassium uptake under osmotic stress. While at low potassium availability, the presence of the KtrAB transporter improves both potassium accumulation and growth of C. glutamicum upon osmotic stress, at proper potassium supply, the channel CglK is sufficient.     

Positively charged mineral surfaces promoted the accumulation of organic intermediates at the origin of metabolism

Identifying plausible mechanisms for compartmentalization and accumulation of the organic intermediates of early metabolic cycles in primitive cells has been a major challenge in theories of life’s origins. Here, we propose a mechanism, where positive membrane potentials elevate the concentration of the organic intermediates. Positive membrane potentials are generated by positively charged surfaces of protocell membranes due to accumulation of transition metals. We find that (i) positive membrane potentials comparable in magnitude to those of modern cells can increase the concentration of the organic intermediates by several orders of magnitude; (ii) generation of large membrane potentials destabilize ion distributions; (iii) violation of electroneutrality is necessary to induce nonzero membrane potentials; and (iv) violation of electroneutrality enhances osmotic pressure and diminishes reaction efficiency, resulting in an evolutionary driving force for the formation of lipid membranes, specialized ion channels, and active transport systems.     

Tn7 elements: engendering diversity from chromosomes to episomes

The bacterial transposon Tn7 maintains two distinct lifestyles, one in horizontally transferred DNA and the other in bacterial chromosomes. Access to these two DNA pools is mediated by two separate target selection pathways. The proteins involved in these pathways have evolved to specifically activate transposition into their cognate target-sites using entirely different recognition mechanisms, but the same core transposition machinery. In this review we discuss how the molecular mechanisms of Tn7-like elements contribute to their diversification and how they affect the evolution of their host genomes. The analysis of over 50 Tn7-like elements provides insight into the evolution of Tn7 and Tn7 relatives. In addition to the genes required for transposition, Tn7-like elements transport a wide variety of genes that contribute to the success of diverse organisms. We propose that by decisively moving between mobile and stationary DNA pools, Tn7-like elements accumulate a broad range of genetic material, providing a selective advantage for diverse host bacteria.     

Propionyl Coenzyme A (Propionyl-CoA) Carboxylase in Haloferax mediterranei: Indispensability for Propionyl-CoA Assimilation and Impacts on Global Metabolism

Propionyl coenzyme A (propionyl-CoA) is an important intermediate during the biosynthesis and catabolism of intracellular carbon storage of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in haloarchaea. However, the haloarchaeal propionyl-CoA carboxylase (PCC) and its physiological significance remain unclear. In this study, we identified a PCC that catalyzed propionyl-CoA carboxylation with an acetyl-CoA carboxylation side activity in Haloferax mediterranei. Gene knockout/complementation demonstrated that the PCC enzyme consisted of a fusion protein of a biotin carboxylase and a biotin-carboxyl carrier protein (PccA [HFX_2490]), a carboxyltransferase component (PccB [HFX_2478]), and an essential small subunit (PccX [HFX_2479]). Knockout of pccBX led to an inability to utilize propionate and a higher intracellular propionyl-CoA level, indicating that the PCC enzyme is indispensable for propionyl-CoA utilization. Interestingly, H. mediterranei DBX (pccBX-deleted strain) displayed multiple phenotypic changes, including retarded cell growth, decreased glucose consumption, impaired PHBV biosynthesis, and wrinkled cells. A propionyl-CoA concentration equivalent to the concentration that accumulated in DBX cells was demonstrated to inhibit succinyl-CoA synthetase of the tricarboxylic acid cycle in vitro. Genome-wide microarray analysis showed that many genes for glycolysis, pyruvate oxidation, PHBV accumulation, electron transport, and stress responses were affected in DBX. This study not only identified the haloarchaeal PCC for the metabolism of propionyl-CoA, an important intermediate in haloarchaea, but also demonstrated that impaired propionyl-CoA metabolism affected global metabolism in H. mediterranei.     

Comparative genomic analysis of the Haloferax volcanii DS2 and Halobacterium salinarium GRB contig maps reveals extensive rearrangement.

Anonymous probes from the genome of Halobacterium salinarium GRB and 12 gene probes were hybridized to the cosmid clones representing the chromosome and plasmids of Halobacterium salinarium GRB and Haloferax volcanii DS2. The order of and pairwise distances between 35 loci uniquely cross-hybridizing to both chromosomes were analyzed in a search for conservation. No conservation between the genomes could be detected at the 15-kbp resolution used in this study. We found distinct sets of low-copy-number repeated sequences in the chromosome and plasmids of Halobacterium salinarium GRB, indicating some degree of partitioning between these replicons. We propose alternative courses for the evolution of the haloarchaeal genome: (i) that the majority of genomic differences that exist between genera came about at the inception of this group or (ii) that the differences have accumulated over the lifetime of the lineage. The strengths and limitations of investigating these models through comparative genomic studies are discussed.     

Culturable epiphytic bacteria isolated from Teleogryllus occipitalus crickets metabolize insecticides

The development of insecticide resistance is attributed to evolutionary changes in pest insect genomes, such as alteration of drug target sites, upregulation of degrading enzymes, and enhancement of drug excretion. Beyond these well‐known mechanisms, symbiotic bacteria may confer insecticide resistance to host crickets. The current study was designed to screen all possible culturable bacterial groups found living in and on the bodies of Teleogryllus occipitalis crickets. We recovered 263 visible bacterial colonies and cultured them individually. After identifying the colonies based on morphology and phylogenetic analysis, we shortlisted 55 bacterial strains belonging to 28 genera. Of these 55 bacterial strains, 18 degraded at least 50% of the original amount of 400 mg/L chlorpyrifos (CP) after 24 hr of coculture. Six of these strains degraded more than 70% of the original amount of 400 mg/L CP. Three strains had antagonistic effects on Bacillus thuringiensis growth. Additionally, the ability of the isolates to degrade glyphosate, phoxim, and esfenvalerate was assessed. We also detected extracellular hydrolase enzyme activities in these isolates. We propose that epiphytic bacterial strains play multiple roles in cricket biology, one of which contributes to chemical and biological pesticide resistance.