Isolation of a member of the candidate phylum 'Atribacteria' reveals a unique cell membrane structure.

A key feature that differentiates prokaryotic cells from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we isolate a member of the ubiquitous, yet-to-be-cultivated phylum 'Candidatus Atribacteria' (also known as OP9) that has an intracytoplasmic membrane apparently surrounding the nucleoid. The isolate, RT761, is a subsurface-derived anaerobic bacterium that appears to have three lipid membrane-like layers, as shown by cryo-electron tomography. Our observations are consistent with a classical gram-negative structure with an additional intracytoplasmic membrane. However, further studies are needed to provide conclusive evidence for this unique intracellular structure. The RT761 genome encodes proteins with features that might be related to the complex cellular structure, including: N-terminal extensions in proteins involved in important processes (such as cell-division protein FtsZ); one of the highest percentages of transmembrane proteins among gram-negative bacteria; and predicted Sec-secreted proteins with unique signal peptides. Physiologically, RT761 primarily produces hydrogen for electron disposal during sugar degradation, and co-cultivation with a hydrogen-scavenging methanogen improves growth. We propose RT761 as a new species, Atribacter laminatus gen. nov. sp. nov. and a new phylum, Atribacterota phy. nov.

Building the bacterial cell wall at the pole.

Polar growth is the predominant mode of cell wall extension in the Actinobacteria and the alphaproteobacterial clade Rhizobiales. The observation of polar elongation in taxonomically diverse bacteria suggests that polar growth may have evolved independently. Indeed, the regulatory mechanisms governing the assembly of cell wall biosynthesis machinery at the pole are distinct in the Actinobacteria and Rhizobiales. Here we highlight recent advances in our understanding of polar growth mechanisms in bacteria, with an emphasis on Streptomyces and Agrobacterium. This review illustrates that common themes are emerging in the regulation of polar growth in diverse bacteria. Emerging themes include the use of landmark proteins to direct growth to the pole and coordination of polar growth with cell-cycle progression.

Comparative genomics of cultured and uncultured strains suggests genes essential for free-living growth of Liberibacter.

The full genomes of two uncultured plant pathogenic Liberibacter, Ca. Liberibacter asiaticus and Ca. Liberibacter solanacearum, are publicly available. Recently, the larger genome of a closely related cultured strain, Liberibacter crescens BT-1, was described. To gain insights into our current inability to culture most Liberibacter, a comparative genomics analysis was done based on the RAST, KEGG, and manual annotations of these three organisms. In addition, pathogenicity genes were examined in all three bacteria. Key deficiencies were identified in Ca. L. asiaticus and Ca. L. solanacearum that might suggest why these organisms have not yet been cultured. Over 100 genes involved in amino acid and vitamin synthesis were annotated exclusively in L. crescens BT-1. However, none of these deficiencies are limiting in the rich media used to date. Other genes exclusive to L. crescens BT-1 include those involved in cell division, the stringent response regulatory pathway, and multiple two component regulatory systems. These results indicate that L. crescens is capable of growth under a much wider range of conditions than the uncultured Liberibacter strains. No outstanding differences were noted in pathogenicity-associated systems, suggesting that L. crescens BT-1 may be a plant pathogen on an as yet unidentified host.

Starch filler and osmoprotectants improve the survival of rhizobacteria in dried alginate beads.

This work deals with optimising the cell survival of rhizobacteria encapsulated in alginate beads filled with starch. Immobilisation of rhizobacteria was done by dripping alginate-starch solution mixed with rhizobacteria into a calcium solution. Beads were analysed based on matrix formulation, bacteria growth phase, osmoprotectants and nature of calcium solution. Maximum cell recovery was obtained on Raoultella terrigena grown in medium supplemented with trehalose and calcium gluconate as gelling agent. Furthermore, dried beads containing Azospirillum brasilense presented 76% of viable cells after one year of storage. The survival of rhizobacteria during the bioencapsulation process can be improved by incorporating starch on beads composition, varying the growth phase of cells and using trehalose in growth culture medium. This work provides a selection of appropriate methods to improve the surviving rate of encapsulated cells during their production and long-term storage (∼1 year at 4°C).

Characterization of indigenous rhizobia from Caatinga

The aim of this study was to characterize rhizobial isolates from Cratylia mollis Mart. ex Benth, Calliandra depauperata Benth. and Mimosa tenuiflora (Willd.) Poir. by means of rhizobial colonies morphology and restriction analysis of the 16S ribosomal gene (16S rDNA-ARDRA). Nodules were collected in the field and from plants cultivated in a greenhouse experiment using Caatinga soil samples. Sixty seven isolates were described by morphological analysis. Forty seven representative isolates were used for ARDRA analysis using seven restriction enzymes. We observed high diversity of both slow and fast-growing rhizobia that formed three morpho-physiological clusters. A few fast-growing isolates formed a group of strains of the Bradyrhizobium type; however, most of them diverged from the B. japonicum and B. elkanii species. Cratylia mollis nodule isolates were the most diverse, while all Mimosa tenuiflora isolates displayed fast growth with no pH change and were clustered into groups bearing 100 percent similarity, according to ARDRA results.

Methylocella tundrae sp. nov., a novel methanotrophic bacterium from acidic tundra peatlands.

A novel species, Methylocella tundrae, is proposed for three methanotrophic strains (T4T, TCh1 and TY1) isolated from acidic Sphagnum tundra peatlands. These strains are aerobic, Gram-negative, non-motile, dinitrogen-fixing rods that possess a soluble methane monooxygenase and utilize the serine pathway for carbon assimilation. Strains T4T, TCh1 and TY1 are moderately acidophilic organisms capable of growth between pH 4.2 and 7.5 (optimum 5.5-6.0) and between 5 and 30 degrees C (optimum 15 degrees C). The major phospholipid fatty acid is 18:1omega7c. The DNA G+C content of strain T4T is 63.3 mol%. The three strains possess almost identical 16S rRNA gene sequences and are most closely related to two previously identified species of Methylocella, Methylocella palustris (97% similarity) and Methylocella silvestris (97.5% similarity). DNA-DNA hybridization values of strain T4T with Methylocella palustris KT and Methylocella silvestris BL2T were respectively 27 and 36%. Thus, the tundra strains represent a novel species, for which the name Methylocella tundrae sp. nov. is proposed. Strain T4T (=DSM 15673T=NCIMB 13949T) is the type strain.

Sucrose is a nonaccumulated osmoprotectant in Sinorhizobium meliloti.

Intracellular accumulation of sucrose in response to lowered water activity seems to occur only in photosynthetic organisms. Here we demonstrate, for the first time, the potent ability of this common sugar, supplied exogenously, to reduce growth inhibition of Sinorhizobium meliloti cells in media of inhibitory osmolarity. Independently of the nature of the growth substrates and the osmotic agent, sucrose appears particularly efficient in promoting the recovery of cytoplasmic volume after plasmolysis. Surprisingly, sucrose is not accumulated by the bacteria at an osmotically efficient level. Instead, it strongly stimulates the accumulation of the main endogenous osmolytes glutamate and N-acetylglutaminylglutamine amide (NAGGN). Examining cell volume changes during the hyperosmotic treatment, we found a close correlation between the enhancement of the osmotically active solute pool and the increase in cell volume. Sucrose shares several features with ectoine, another nonaccumulated osmoprotectant for S. meliloti. Overall, osmoregulation in S. meliloti appears to be strongly divergent from that in most bacteria.

Interactions between heterologous FtsA and FtsZ proteins at the FtsZ ring.

FtsZ and FtsA are essential for cell division in Escherichia coli and colocalize to the septal ring. One approach to determine what regions of FtsA and FtsZ are important for their interaction is to identify in vivo interactions between FtsA and FtsZ from different species. As a first step, the ftsA genes of Rhizobium meliloti and Agrobacterium tumefaciens were isolated and characterized. In addition, an FtsZ homolog that shared the unusual C-terminal extension of R. meliloti FtsZ1 was found in A. tumefaciens. In order to visualize their localization in cells, we tagged these proteins with green fluorescent protein (GFP). When R. meliloti FtsZ1-GFP or A. tumefaciens FtsZ-GFP was expressed at low levels in E. coli, they specifically localized only to the E. coli FtsZ ring, possibly by coassembly. When A. tumefaciens FtsA-GFP or R. meliloti FtsA-GFP was expressed in E. coli, they failed to localize detectably to the E. coli FtsZ ring. However, when R. meliloti FtsZ1 was coexpressed with them, fluorescence localized to a band at the midcell division site, strongly suggesting that FtsA from either A. tumefaciens or R. meliloti can bind directly to its cognate FtsZ. As expected, GFP-tagged FtsZ1 and FtsA from either R. meliloti or A. tumefaciens localized to the division site in A. tumefaciens cells. Therefore, the 61 amino acid changes between A. tumefaciens FtsA and R. meliloti FtsA do not prevent their direct interaction with FtsZ1 from either species, suggesting that those residues are not essential for protein-protein contacts. Moreover, the failure of the two non-E. coli FtsA derivatives to interact strongly with E. coli FtsZ in this in vivo system unless their cognate FtsZ was also present suggests that FtsA-FtsZ interactions have coevolved and that the residues which differ between the E. coli proteins and those of the two other species may be important for specific interactions.

In vivo nuclear magnetic resonance study of the osmoregulation of phosphocholine-substituted beta-1,3;1,6 cyclic glucan and its associated carbon metabolism in Bradyrhizobium japonicum USDA 110.

A phosphocholine-substituted beta-1,3;1,6 cyclic glucan (PCCG), an unusual cyclic oligosaccharide, has been isolated from Bradyrhizobium japonicum USDA 110 (D. B. Rolin, P. E. Pfeffer, S. F. Osman, B. S. Swergold, F. Kappler, and A. J. Benesi, Biochim. Biophys. Acta 1116:215-225, 1992). Data presented here suggest that PCCG synthesis is dependent on the carbon metabolism and that osmotic regulation of its biosynthesis parallels regulation of membrane-derived oligosaccharide biosynthesis observed in Escherichia coli (E. P. Kennedy, M. K. Rumley, H. Schulman, and L. M. G. van Golde, J. Biol. Chem. 251:4208-4213, 1976) and Agrobacterium tumefaciens (G. A. Cangelosi, G. Martinetti, and E. W. Nester, J. Bacteriol. 172:2172-2174, 1990). Growth of B. japonicum USDA 110 cells in the reference medium at relatively low osmotic pressures (LO) (65 mosmol/kg of H2O) caused a large accumulation of PCCG and unsubstituted beta-1,3;1,6 cyclic glucans (CG). Sucrose and polyethylene glycol, nonionic osmotica, reduce all growth rates and inhibit almost completely the production of PCCG at high osmotic pressures (HO) above 650 and 400 mosmol/kg of H2O), respectively. We used in vivo 13C nuclear magnetic resonance spectroscopy to identify the active osmolytes implicated in the osmoregulation process. The level of alpha,alpha-trehalose in B. japonicum cells grown in autoclaved or filter-sterilized solutions remained constant in HO (0.3 M sucrose or 250 g of polyethylene glycol 6000 per liter) medium. Significant amounts of glycogen and extracellular polysaccharides were produced only when glucose was present in the autoclaved HO 0.3 M sucrose media. The results of hypo- and hyperosmotic shocking of B. japonicum USDA 110 cells were monitored by using in vivo 31P and 13C nuclear magnetic resonance spectroscopy. The first observed osmoregulatory response of glycogen-containing cells undergoing hypoosmotic shock was release of P(i) into the medium. Within 7 h, reabsorption of P(i) was complete and production of PCCG was initiated. After 12 h, the PCCG content had increased by a factor of 7. Following the same treatment, cells containing little or no glycogen released trehalose and failed to produce PCCG. Thus the production of PCCG/CG in response to hypoosmotic shocking of stationary-phase cells was found to be directly linked to the interconversion of stored glycogen. Hyperosmotic shocking of LO-grown stationary-phase cells with sucrose had no effect on the content of previously synthesized CG/PCCG. The PCCG/CG content and its osmotically induced biosynthesis are discussed in terms of carbon metabolism and a possible role in hypoosmotic adaptation in B. japonicum USDA 110.

Polarized cells, polar actions.

The recognition of polar bacterial organization is just emerging. The examples of polar localization given here are from a variety of bacterial species and concern a disparate array of cellular functions. A number of well-characterized instances of polar localization of bacterial proteins, including the chemoreceptor complex in both C. crescentus and E. coli, the maltose-binding protein in E. coli, the B. japonicum surface attachment proteins, and the actin tail of L. monocytogenes within a mammalian cell, involve proteins or protein complexes that facilitate bacterial interaction with the environment, either the extracellular milieux or that within a plant or mammalian host. The significance of this observation remains unclear. Polarity in bacteria poses many problems, including the necessity for a mechanism for asymmetrically distributing proteins as well as a mechanism by which polar localization is maintained. Large structures, such as a flagellum, are anchored at the pole by means of the basal body that traverses the peptidoglycan wall. But for proteins and small complexes, whether in the periplasm or the membrane, one must invoke a mechanism that prevents the diffusion of these proteins away from the cell pole. Perhaps the periplasmic proteins are retained at the pole by the presence of the periseptal annulus (35). The constraining features for membrane components are not known. For large aggregates, such as the clusters of MCP, CheA, and CheW complexes, perhaps the size of the aggregate alone prevents displacement. In most cases of cellular asymmetry, bacteria are able to discriminate between the new pole and the old pole and to utilize this information for localization specificity. The maturation of new pole to old pole appears to be a common theme as well. Given numerous examples reported thus far, we propose that bacterial polarity displays specific rules and is a more general phenomenon than has been previously recognized.

Carbohydrate binding activities of Bradyrhizobium japonicum: unipolar localization of the lectin BJ38 on the bacterial cell surface.

A polyclonal antiserum generated against the Bradyrhizobium japonicum lectin BJ38 was characterized to be specifically directed against the protein. Treatment of B. japonicum cells with this antiserum and subsequent visualization with transmission electron microscopy and both conventional and confocal fluorescence microscopy revealed BJ38 at only one pole of the bacterium. BJ38 appeared to be organized in a tuft-like mass, separated from the bacterial outer membrane. BJ38 localization was coincident with the attachment site for (i) homotypic agglutination to other B. japonicum cells, (ii) adhesion to the cultured soybean cell line SB-1, and (iii) adsorption to Sepharose beads covalently derivatized with lactose. In contrast, the plant lectin soybean agglutinin labeled the bacteria at the pole distant from the bacterial attachment site. These results indicate that the topological distribution of BJ38 is consistent with a suggested role for this bacterial lectin in the polar binding of B. japonicum to other cells and surfaces.