Cell envelopes of chemotaxis mutants of Escherichia coli rotate their flagella counterclockwise.

Flagella rotated exclusively counterclockwise in Escherichia coli cell envelopes prepared from wild-type cells, whose flagella rotated both clockwise and counterclockwise, from mutants rotating their flagella counterclockwise only, and even from mutants rotating their flagella primarily clockwise. Some factor needed for clockwise flagellar rotation appeared to be missing or defective in the cell envelopes.     

Structure of the intracellular part of the motility apparatus of halobacteria

he electron microscopic study of the structure of the motility apparatus of the archaea Halobacterium salinarium 4W12 and Natronobacterium magadii confirmed our earlier observation that the motility apparatus of halobacteria contains an intracellular disk-shaped lamellar structure (DLS). Polar cap structures (PCSs) isolated from the halobacterium were preliminarily identified as the DLSs. The PCSs in complexes with flagella were also isolated from the haloalkaliphilic bacterium N. magadii. The specific structure of the archaeal motility apparatus is discussed.     

In vitro Approach to Bacterial Chemotaxis

An in vitro approach to study bacterial motility and chemotaxis is described. The approach is based on a preparation of flagellated cell envelopes. The envelopes are prepared from bacteria by a penicillin treatment and subsequent osmotic lysis. When the envelopes are energized, their flagella rotate. The direction of rotation in wild type envelopes is counterclockwise. Inclusion of the CheY protein within the envelopes may restore clockwise rotation. The advantages and disadvantages of this approach are pointed out.     

Minimal requirements for rotation of bacterial flagella.

An in vitro system of cell envelopes from Salmonella typhimurium with functional flagella was used to determine the minimal requirements for flagellar rotation. Rotation in the absence of cytoplasmic constituents could be driven either by respiration or by an artificially imposed chemical gradient of protons. No specific ionic requirements other than protons (or hydroxyls) were found for the motor function.     

New structural features of the flagellar base in Salmonella typhimurium revealed by rapid-freeze electron microscopy.

The structure of the flagellar base in Salmonella typhimurium has been studied by rapid-freeze techniques. Freeze-substituted thin sections and freeze-etched replicas of cell envelope preparations have provided complementary information about the flagellar base. The flagellar base has a bell-shaped extension reaching as far as 50 nm into the bacterial cytoplasm. This structure can be recognized in intact bacteria but was studied in detail in cell envelopes, where some flagella lacking parts of the bell were helpful in understanding its substructure. Structural relationships may be inferred between this cytoplasmic component of the flagellum and the recently described flagellar intramembrane particle rings as well as the structures associated with the basal body in isolated, chemically fixed flagella.     

Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium

Dundas, Ian E. D. (University of Illinois, Urbana), and H. Orin Halvorson. Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium. J. Bacteriol. 91:113-119. 1966.-Arginine was shown to be essential for growth of Halobacterium salinarium strain 1 in a chemically defined medium. Citrulline was the only compound which could substitute for arginine without affecting growth. Resting cells of H. salinarium converted arginine to citrulline and citrulline to ornithine. Cells grown in an arginine-free medium with C(14)-ureido-labeled citrulline incorporated the isotope mainly into the arginine of their proteins. The enzymes arginine desimidase and ornithine transcarbamylase were found and studied in cell-free extracts of H. salinarium. Experiments indicated that arginine was degraded in H. salinarium by arginine desimidase to citrulline, and that citrulline was further degraded by ornithine transcarbamylase to carbamyl phosphate and ornithine. Synthesis of arginine from citrulline seems to occur via the formation of argininosuccinic acid.     

The specificity of fumarate as a switching factor of the bacterial flagellar motor

Fumarate restores to flagella of cytoplasm-free, CheY- containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemo-taxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.     

Direction of flagellar rotation in bacterial cell envelopes

Cell envelopes with functional flagella, isolated from wild-type strains of Escherichia coli and Salmonella typhimurium by formation of spheroplasts with penicillin and subsequent osmotic lysis, demonstrate counterclockwise (CCW)-biased rotation when energized with an electron donor for respiration, DL-lactate. Since the direction of flagellar rotation in bacteria is central to the expression of chemotaxis, we studied the cause of this bias. Our main observations were: (i) spheroplasts acquired a clockwise (CW) bias if instead of being lysed they were further incubated with penicillin; (ii) repellents temporarily caused CW rotation of tethered bacteria and spheroplasts but not of their derived cell envelopes; (iii) deenergizing CW-rotating cheV bacteria by KCN or arsenate treatment caused CCW bias; (iv) cell envelopes isolated from CW-rotating cheC and cheV mutants retained the CW bias, unlike envelopes isolated from cheB and cheZ mutants, which upon cytoplasmic release lost this bias and acquired CCW bias; and (v) an inwardly directed, artificially induced proton current rotated tethered envelopes in CCW direction, but an outwardly directed current was unable to rotate the envelopes. It is concluded that (i) a cytoplasmic constituent is required for the expression of CW rotation (or repression of CCW rotation) in strains which are not defective in the switch; (ii) in the absence of this cytoplasmic constituent, the motor is not reversible in such strains, and it probably is mechanically constricted so as to permit CCW sense of rotation only; (iii) the requirement of CW rotation for ATP is not at the level of the motor or the switch but at one of the preceding functional steps of the chemotaxis machinery; (iv) the cheC and cheV gene products are associated with the cytoplasmic membrane; and (v) direct interaction between the switch-motor system and the repellent sensors is improbable.     

Correlation between phosphorylation of the chemotaxis protein CheY and its activity at the flagellar motor.

Phosphorylation of the chemotaxis protein CheY by its kinase CheA appears to play a central role in the process of signal transduction in bacterial chemotaxis. It is presumed that the role is activation of CheY which results in clockwise (CW) flagellar rotation. The aim of this study was to determine whether this activity of CheY indeed depends on the protein being phosphorylated. Since the phosphorylation of CheY can be detected only in vitro, we studied the ability of CheY to cause CW rotation in an in vitro system, consisting of cytoplasm-free envelopes of Salmonella typhimurium or Escherichia coli having functional flagella. Envelopes containing just buffer rotated only counterclockwise. Inclusion of CheY caused 14% of the rotating envelopes to go CW. This fraction of CW-rotating envelopes was not altered when the phosphate potential in the envelopes was lowered by inclusion of ADP together with CheY in them, indicating that CheY has a certain degree of activity even without being phosphorylated. Attempts to increase the activity of CheY in the envelopes by phosphorylation were not successful. However, when CheY was inserted into partially-lysed cells (semienvelopes) under phosphorylating conditions, the number of CW-rotating cells increased 3-fold. This corresponds to more than a 100-fold increase in the activity of a single CheY molecule upon phosphorylation. It is concluded that nonphosphorylated CheY can interact with the flagellar switch and cause CW rotation, but that this activity is increased by at least 2 orders of magnitude by phosphorylation. This increase in activity requires additional cytoplasmic constituents, the identity of which is not yet known.     

Isolation of an ftsZ homolog from the archaebacterium Halobacterium salinarium: implications for the evolution of FtsZ and tubulin.

We have isolated a homolog of the cell division gene ftsZ from the extremely halophilic archaebacterium Halobacterium salinarium. The predicted protein of 39 kDa is divergent relative to eubacterial homologs, with 32% identity to Escherichia coli FtsZ. No other eubacterial cell division gene homologs were found adjacent to H. salinarium ftsZ. Expression of the ftsZ gene region in H. salinarium induced significant morphological changes leading to the loss of rod shape. Phylogenetic analysis demonstrated that the H. salinarium FtsZ protein is more related to tubulins than are the FtsZ proteins of eubacteria, supporting the hypothesis that FtsZ may have evolved into eukaryotic tubulin.     

Antigenic Behaviors of Two Axial Flagellar Proteins Detected in Treponema denticola

Two polypeptide antigens with molecular sizes of 34,000 daltons (34 kDa) and 38 kDa were separated from heated cells of a human clinical treponeme strain G7201 and Treponema denticola ATCC 35404, respectively. The rabbit polyclonal antisera against these antigens were produced and examined for their immunological reactions with the two heated antigens or intact spirochetal cells. Immunoblot analysis showed that the 34-kDa protein was also detected in T. denticola ATCC 35404 and ATCC 33520, and the 38-kDa protein was detected only in the two ATCC strains. Immunoelectron microscopy using the two rabbit antisera and protein A-gold complexes demonstrated that the 38-kDa protein antigen was present on the axial flagella of two T. denticola strains, and that the 34-kDa protein was located in the axial flagella of the G7201 cell, but neither in axial flagella nor on outer envelopes of the two ATCC strains cells, suggesting that the native 34-kDa axial flagellar protein of the G7201 strain may be different from that of T. denticola in terms of immunological reactivity.     

Lectin binding in Cryptomonas and Chroomonas (Cryptophyceae)

The cell envelopes of Cryptomonas and Chroomonas exhibited significant fluorescence using FITC-labelled concanavalin A and wheat germ agglutinin when the cells were fixed prior to lectin binding. The periplast became intensely labelled in Chroomonas whereas Cryptomonas showed fluorescing granula in its gullet/furrow region and on the cell surface. Lectin labelling followed by fixation showed only label of periplast remnants of lysed cells and of the flagella of Chroomonas. Isolated periplasts of Cryptomonas and Chroomonas were intensively labelled with both concanavalin A and wheat germ agglutinin. Glycostaining of gels, onto which total cell protein extracts were loaded, showed a glycoprotein of high molecular weight for Cryptomonas and Chroomonas and an additional glycoprotein for Cryptomonas species.     

Ketocarotenoids in halobacteria: 3-hydroxy-echinenone and trans-astaxanthin

HPLC testing of the carotenoid content of the halobacteria Halobacterium salinarium, Haloarcula hispanica and Haloferax mediterranei showed that all contained high amounts of ketocarotenoids. Halobacterium salinarium produced 2400 μg of total carotenoids per gram of dried bacteria, including 265 μg of trans -astaxanthin (11%), and 588 μg of 3-hydroxy-echinenone (24%). The biotechnological properties of Halobacterium salinarium as a natural pigment source are also presented. The results are compared with those from the yeast Phaffia rhodozyma currently used in the industry as a source of trans -astaxanthin.     

Inorganic polyphosphates and phosphohydrolases from Halobacterium salinarium

Halobacterium salinarium grown in a liquid medium consumed up to 75% of phosphates originally present in the growth medium and accumulated up to 100 mumol Pi/g wet biomass by the time it entered the growth retardation phase. The content of acid-soluble oligophosphates in the biomass was maximum at the early stage of active growth and drastically decreased when cells reached the growth-retardation phase. The total content of alkali-soluble and acid-insoluble polyphosphates changed very little throughout the cultivation period (five days). The polyphosphate content of H. salinarium cells was close to that of yeasts and eubacteria. The pyrophosphatase, polyphosphatase, and nonspecific phosphatase activities of H. salinarium cells were several times lower than those of the majority of eubacteria. The specific activity of pyrophosphatase, the most active hydrolase of H. salinarium, gradually increased during cultivation, reaching 540 mU/mg protein by the end of the cultivation period. Half of the total pyrophosphatase activity of this halobacterium was localized in the cytosol. The molecular weight of pyrophosphatase, evaluated by gel filtration, was 86 kDa. The effective Km of this enzyme with respect to pyrophosphate was 115 microM.     

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.     

Formation of insoluble magnesium phosphates during growth of the archaea Halorubrum distributum and Halobacterium salinarium and the bacterium Brevibacterium antiquum

Stationary phase cells of the halophilic archaea Halobacterium salinarium and Halorubrum distributum, growing at 3-4 M NaCl, and of the halotolerant bacterium Brevibacterium antiquum, growing with and without 2.6 NaCl, took up approximately 90% of the phosphate from the culture media containing 2.3 and 11.5 mM phosphate. The uptake was blocked by the uncoupler FCCP. In B. antiquum, EDTA inhibited the phosphate uptake. The content of polyphosphates in the cells was significantly lower than the content of orthophosphate. At a high phosphate concentration, up to 80% of the phosphate taken up from the culture medium was accumulated as Mg(2)PO(4)OH x 4H(2)O in H. salinarium and H. distributum and as NH(4)MgPO(4) x 6H(2)O in B. antiquum. Consolidation of the cytoplasm and enlargement of the nucleoid zone were observed in the cells during phosphate accumulation. At phosphate surplus, part of the H. salinarium and H. distributum cell population was lysed. The cells of B. antiquum were not lysed and phosphate crystals were observed in the cytoplasm.     

Chemotaxis and phototaxis require a CheA histidine kinase in the archaeon Halobacterium salinarium.

Histidine kinases are part of the two-component signal transduction system responsible for eubacterial responses to diverse environmental signals. They have recently been detected in eukaryotes but their existence in the kingdom Archaea remains uncertain. Here we report the sequence and function of a histidine kinase (CheAH.s.) from Halobacterium salinarium, the first such transmitter in Archaea. The protein CheAH.s. (668 residues) has significant sequence identity with the CheA proteins known from eubacterial signal transduction (e.g. 34% identity with CheA from Bacillus subtilis). Antibodies were raised against CheAH.s. as expressed in Escherichia coli and were used in Western blotting to demonstrate the expression of cheAH.s. in H. salinarium. As has been observed for other halophilic proteins, CheAH.s. has a deviant electrophoretic migration, with an apparent molecular weight of 103 kDa on SDS-PAGE compared with a calculated molecular weight of 72 kDa. Deletion of a part of the cheAH.s. gene leads to loss of both chemotactic and phototactic responses in H. salinarium as measured by swarm plate assays, motion analysis and tethering experiments. This indicates that CheAH.s. plays a crucial role in chemical and light signal integration, presumably interacting with at least two phototransducers and a number of chemoreceptors.     

Acetyladenylate or its derivative acetylates the chemotaxis protein CheY in vitro and increases its activity at the flagellar switch.

CheY, a key protein in the mechanism of bacterial chemotaxis, is known to interact with the flagellar switch and thereby cause clockwise rotation. This activity of CheY was significantly increased by producing acetyladenylate (AcAMP) within cytoplasm-free bacterial envelopes containing purified CheY. This was achieved by including in the envelopes the enzyme acetyl-CoA synthetase (ACS) and ATP, and adding acetate externally. The fraction of clockwise-rotating envelopes, tethered to glass by their flagella, increased from 14% to 58% by the presence of AcAMP (or its derivative). In parallel experiments carried out with [14C]acetate under similar conditions, CheY became acetylated: [1-14C]acetate was as effective as [2-14C]acetate in labeling CheY, and ACS-dependent labeling of CheY by [alpha-32P]ATP was not detected. The switch proteins, FliG, FliM, and FliN, isolated to purity, were not acetylated. The acetylation was specific for CheY and dependent on its native conformation. The acetylated form the CheY was estimated to be more active than its nonacetylated form by 4-5 orders of magnitude. Acetylated CheY was stable in the presence of the strong nucleophiles hydroxylamine or ethanolamine, indicative of N-acetylation. There was a correlation between the activity of CheY in vivo and its ability to be acetylated in vitro. Thus, proteins with a single substitution at their active site, CheY57DE and CheY109KR, are not active in vivo and accordingly were not acetylated in vitro; in contrast, the protein CheY13DK is active in vivo and was normally acetylated in vitro. The possibility that CheY acetylation plays a role in bacterial chemotaxis is discussed.     

Isolation and characterization of an extremely halophilic archaeobacterium from traditionally fermented Thai fish sauce (nam pla)

An extremely halophilic archaeobacterium (halobacterium), strain ORE, was isolated from traditionally fermented Thai fish sauce (nam pla), which has a concentration of 4·4–5·1 M NaCl. Polar liquid analysis and DNA hybridization revealed that it was a representative of the species Halobacterium salinarium. In common with many other strains of H. salinarium this organism produces salt-stable extracellular proteases which are likely to be important in the fermentation process.