Transformations in flagellar structure of Rhodobacter sphaeroides and possible relationship to changes in swimming speed

Rhodobacter sphaeroides is a photosynthetic bacterium which swims by rotating a single flagellum in one direction, periodically stopping, and reorienting during these stops. Free-swimming R. sphaeroides was examined by both differential interference contrast (DIC) microscopy, which allows the flagella of swimming cells to be seen in vivo, and tracking microscopy, which tracks swimming patterns in three dimensions. DIC microscopy showed that when rotation stopped, the helical flagellum relaxed into a high-amplitude, short-wavelength coiled form, confirming previous observations. However, DIC microscopy also revealed that the coiled filament could rotate slowly, reorienting the cell before a transition back to the functional helix. The time taken to reform a functional helix depended on the rate of rotation of the helix and the length of the filament. In addition to these coiled and helical forms, a third conformation was observed: a rapidly rotating, apparently straight form. This form took shape from the cell body out and was seen to form directly from flagella that were initially in either the coiled or the helical conformation. This form was always significantly longer than the coiled or helical form from which it was derived. The resolution of DIC microscopy made it impossible to identify whether this form was genuinely in a straight conformation or was a low-amplitude, long-wavelength helix. Examination of the three-dimensional swimming pattern showed that R. sphaeroides changed speed while swimming, sometimes doubling the swimming speed between stops. The rate of acceleration out of stops was also variable. The transformations in waveform are assumed to be torsionally driven and may be related to the changes in speed measured in free-swimming cells. The roles of and mechanisms that may be involved in the transformations of filament conformations and changes in swimming speed are discussed.     

Real time computer tracking of free-swimming and tethered rotating cells

A computerized image processing system has been developed that tracks individual free-swimming cells and rotating bacterial cell bodies tethered by their flagella in real time. Free-swimming bacteria of Rhodobacter sphaeroides, Rhodospirullum rubrum, and Salmonella typhimurium have been tracked swimming at speeds from 0 to over 120 microns s-1. A high level of discrimination is exerted against noncellular objects, allowing analysis of stopped as well as moving cells. This enabled detection of both speed and qualitative change in the swimming patterns of R. sphaeroides WS8 upon tactic stimulation. Comparison with darkfield microscopy indicated that the two techniques were in substantial agreement. The unidirectional rotation of cells of R. sphaeroides WS8 could be detected when the cells were either parallel to the microscope slide or end on. Frequencies of rotation of up to 10 Hz were monitored before image blurring became a problem. True rods would be easier to analyze at higher speeds of rotation. Although developed for photosynthetic bacteria, a wide range of bacteria, eucaryotic organisms, and subcellular organelles could be tracked with this system. Minor modifications to the software allow customization to different types of motility analysis.     

Response kinetics of tethered bacteria to stepwise changes in nutrient concentration

We examined the changes in swimming behaviour of the bacterium Rhodobacter sphaeroides in response to stepwise changes in a nutrient (propionate), following the pre-stimulus motion, the initial response and the adaptation to the sustained concentration of the chemical. This was carried out by tethering motile cells by their flagella to glass slides and following the rotational behaviour of their cell bodies in response to the nutrient change. Computerised motion analysis was used to analyse the behaviour. Distributions of run and stop times were obtained from rotation data for tethered cells. Exponential and Weibull fits for these distributions, and variability in individual responses are discussed. In terms of parameters derived from the run and stop time distributions, we compare the responses to stepwise changes in the nutrient concentration and the long-term behaviour of 84 cells under 12 propionate concentration levels from 1 nM to 25 mM. We discuss traditional assumptions for the random walk approximation to bacterial swimming and compare them with the observed R. sphaeroides motile behaviour.     

Natural assemblages of marine bacteria exhibiting high-speed motility and large accelerations.

Natural communities of marine bacteria, an isolate (FMB-Bf3) from one marine community, and Escherichia coli were examined by video microscopy for the magnitude and uniformity of their speed. Natural communities formed tight microswarms that showed higher speeds (mean = 230 microns s-1) than did E. coli (15 microns s-1) or FMB-Bf3 (mean = 62 microns s-1). Outside the microswarms, the marine bacteria slowed to 45 microns s-1. Between turns, in mid run, and while travelling in straight lines, the natural-community bacteria accelerated up to 1,450 microns s-2 while the cultured bacteria showed maximum accelerations of 70 and 166 microns s-2. The frequency distribution of speed change for the marine bacteria was skewed towards a few large negative accelerations and a range of positive accelerations. The general pattern was one of relatively slow increases in speed followed by abrupt declines. The results indicate that the mechanical generation and energetic maintenance, as well as the environmental function, of bacterial motility need reappraisal. We conclude that the standard bacterial motility parameters of low and uniform speed, derived from culture-based studies, are not necessarily applicable to marine bacterial communities.     

The chemokinetic and chemotactic behavior of Rhodobacter sphaeroides: two independent responses.

Rhodobacter sphaeroides exhibits two behavioral responses when exposed to some compounds: (i) a chemotactic response that results in accumulation and (ii) a sustained increase in swimming speed. This latter chemokinetic response occurs without any apparent long-term change in the size of the electrochemical proton gradient. The results presented here show that the chemokinetic response is separate from the chemotactic response, although some compounds can induce both responses. Compounds that caused only chemokinesis induced a sustained increase in the rate of flagellar rotation, but chemoeffectors which were also chemotactic caused an additional short-term change in both the stopping frequency and the duration of stops and runs. The response to a change in chemoattractant concentration was a transient increase in the stopping frequency when the concentration was reduced, with adaptation taking between 10 and 60 s. There was also a decrease in the stopping frequency when the concentration was increased, but adaptation took up to 60 min. The nature and duration of both the chemotactic and chemokinetic responses were concentration dependent. Weak organic acids elicited the strongest chemokinetic responses, and although many also caused chemotaxis, there were conditions under which chemokinesis occurred in the absence of chemotaxis. The transportable succinate analog malonate caused chemokinesis but not chemotaxis, as did acetate when added to a mutant able to transport but not grow on acetate. Chemokinesis also occurred after incubation with arsenate, conditions under which chemotaxis was lost, indicating that phosphorylation at some level may have a role in chemotaxis. Aspartate was the only chemoattractant amino acid to cause chemokinesis. Glutamate caused chemotaxis but not chemokinesis. These data suggest that (i) chemotaxis and chemokinesis are separate responses, (ii) metabolism is required for chemotaxis but not chemokinesis, (iii) a reduction in chemoattractant concentration may cause the major chemotactic signal, and (iv) a specific transport pathway(s) may be involved in chemokinetic signalling in R. sphaeroides.     

Motility, chemokinesis, and methylation-independent chemotaxis in Azospirillum brasilense.

Observations of free-swimming and antibody-tethered Azospirillum brasilense cells showed that their polar flagella could rotate in both clockwise and counterclockwise directions. Rotation in a counterclockwise direction caused forward movement of free-swimming cells, whereas the occasional change in the direction of rotation to clockwise caused a brief reversal in swimming direction. The addition of a metabolizable chemoattractant, e.g., malate or proline, had two distinct effects on the swimming behavior of the bacteria: (i) a short-term decrease in reversal frequency from 0.33 to 0.17 s-1 and (ii) a long-term increase in the mean population swimming speed from 13 to 23 microns s-1. A. brasilense therefore shows both chemotaxis and chemokinesis in response to temporal gradients of some chemoeffectors. Chemokinesis was dependent on the growth state of the cells and may depend on an increase in the electrochemical proton gradient above a saturation threshold. Analysis of behavior of a methionine auxotroph, assays of in vivo methylation, and the use of specific antibodies raised against the sensory transducer protein Tar of Escherichia coli all failed to demonstrate the methylation-dependent pathway for chemotaxis in A. brasilense. The range of chemicals to which A. brasilense shows chemotaxis and the lack of true repellents indicate an alternative chemosensory pathway probably based on metabolism of chemoeffectors.     

Permeability of ammonia and amines in Rhodobacter sphaeroides and Bacillus firmus

Permeabilities of uncharged ammonia (NH3), methylamine (CH3NH2), and ethylamine (CH3CH2NH2) in the gram-negative phototrophic bacterium Rhodobacter sphaeroides were measured directly in cells grown heterotrophically under aerobic conditions. The permeability of NH3 was 2.55 +/- 0.73 microns s-1 (n = 20), but the permeabilities of CH3NH2 (MA) and CH3CH2NH2 (EA) were higher, PMA = 17.8 +/- 2.8 microns s-1 (n = 50), PEA = 24.7 +/- 3.9 microns s-1 (n = 44). The relative permeabilities of amines were also determined from their effect on the pH gradient across the cell membrane at alkaline external pH. In aerobically grown R. sphaeroides, both techniques indicated that the permeability of CH3CH2NH2 was about 30% greater than that of CH3NH2 but that the permeability of NH3 was only about 1/5 that of CH3NH2. The relative permeabilities of NH3 (A) and CH3NH2 were different in R. sphaeroides cells grown under three different physiological conditions: (a) cells grown aerobically with ammonium sulfate (PA/PMA about 0.20), (b) cells grown anaerobically with ammonium sulfate as their nitrogen source (PA/PMA about 0.29), and (c) diazotrophic cells (PA/PMA about 0.38). NH3 was also found to be only about 1/3 as permeable as CH3NH2 in the alkalophilic gram-positive bacterium Bacillus firmus. The findings that permeability properties of NH3 and CH3NH2 are very different in different bacteria and vary according to the conditions under which the organism is grown need to be taken into account in the interpretation of experiments where [14C]methylamine is used as an ammonia analog.     

Mechanisms of growth inhibition by propionate and restoration of the growth by sodium bicarbonate or acetate in Rhodopseudomonas sphaeroides S

Mechanisms of growth inhibition by propionate on the growth of Rhodopseudomonas sphaeroides were studied. Partially purified pyruvate dehydrogenase complex (PDC) from R. sphaeroides was inhibited by propionyl-CoA, one of the metabolic intermediates of propionate, while propionate itself did not inhibit the enzyme. This suggests that the inhibitor of the growth in vivo is not propionate but propionyl-CoA. The inhibition by propionyl-CoA was competitive with respect to coenzyme A concentration. The K1 value for propionyl-CoA was 0.84 mM. Addition of NaHCO3, which restored the growth of this bacterium in the presence of propionate, increased the rate of propionate incorporation by 1.7-fold and decreased the intracellular level of propionyl-CoA by half. These findings suggest that HCO3-ion lowers the level of propionyl-CoA by accelerating its carboxylation reaction, which is catalyzed by propionyl-CoA carboxylase. Effects of NaHCO3 and acetate on the growth restoration were also studied by the use of propionyl-CoA carboxylase-deficient mutants. NaHCO3 did not restore the growth of the mutants, indicating an essential role of propionyl-CoA carboxylase on the restoration of growth by NaHCO3 as suggested above. Addition of acetate restores the growth of the mutants in the presence of propionate. Acetate probably restores the growth by supplying acetyl-CoA.     

Rapid bacterial swimming measured in swarming cells of Thiovulum majus.

Swarming cells of the sulfide-oxidizing bacterium Thiovulum majus form bands and show bioconvective patterns of swimming when placed in vessels containing H2S/O2 interfaces. Measurements of swimming velocities with video microscopic recordings under such conditions showed mean cell speeds as high as 615 microns s-1, unprecedented in bacteria.     

Rhizobium meliloti swims by unidirectional, intermittent rotation of right-handed flagellar helices.

The 5 to 10 peritrichously inserted complex flagella of Rhizobium meliloti MVII-1 were found to form right-handed flagellar bundles. Bacteria swam at speeds up to 60 microns/s, their random three-dimensional walk consisting of straight runs and quick directional changes (turns) without the vigorous angular motion (tumbling) seen in swimming Escherichia coli cells. Observations of R. meliloti cells tethered by a single flagellar filament revealed that flagellar rotation was exclusively clockwise, interrupted by very brief stops (shorter than 0.1 s), typically every 1 to 2 s. Swimming bacteria responded to chemotactic stimuli by extending their runs, and tethered bacteria responded by prolonged intervals of clockwise rotation. Moreover, the motility tracks of a generally nonchemotactic ("smooth") mutant consisted of long runs without sharp turns, and tethered mutant cells showed continuous clockwise rotation without detectable stops. These observations suggested that the runs of swimming cells correspond to clockwise flagellar rotation, and the turns correspond to the brief rotation stops. We propose that single rotating flagella (depending on their insertion point on the rod-shaped bacterial surface) can reorient a swimming cell whenever the majority of flagellar motors stop.     

Response kinetics of tethered Rhodobacter sphaeroides to changes in light intensity

Rhodobacter sphaeroides can swim toward a wide range of attractants (a process known as taxis), propelled by a single rotating flagellum. The reversals of motor direction that cause tumbles in Eschericia coli taxis are replaced by brief motor stops, and taxis is controlled by a complex sensory system with multiple homologues of the E. coli sensory proteins. We tethered photosynthetically grown cells of R. sphaeroides by their flagella and measured the response of the flagellar motor to changes in light intensity. The unstimulated bias (probability of not being stopped) was significantly larger than the bias of tethered E. coli but similar to the probability of not tumbling in swimming E. coli. Otherwise, the step and impulse responses were the same as those of tethered E. coli to chemical attractants. This indicates that the single motor and multiple sensory signaling pathways in R. sphaeroides generate the same swimming response as several motors and a single pathway in E. coli, and that the response of the single motor is directly observable in the swimming pattern. Photo-responses were larger in the presence of cyanide or the uncoupler carbonyl cyanide 4-trifluoromethoxyphenylhydrazone (FCCP), consistent with the photo-response being detected via changes in the rate of electron transport.     

Movement of actin away from the center of reconstituted rabbit myosin filament is slower than in the opposite direction.

By decreasing ionic strength slowly, thick filaments of several micrometers in length were obtained from purified rabbit skeletal muscle myosin. Dark-field observation showed these filaments with their center scattering light extensively. Active movement of actin filaments complexed with tetramethyl rhodamine-phalloidin along the reconstituted myosin filaments was observed. Actin filaments moved towards the center of myosin filaments at a speed of 3.9 +/- 1.6 microns s-1 (mean +/- SD, n = 40) and often continued to move beyond the center towards the tip of the opposite side at a lower speed. The speed of the movement away from the center was 1.0 +/- 0.6 microns s-1 (n = 59). Thus, the functional bipolarity in terms of the movement speed which was first found in native thick filaments of molluscan smooth muscle is also seen in reconstituted filaments from purified rabbit skeletal muscle myosin. The difference of the speed between the two directions is considered to be due to properties of myosin molecules themselves.     

The flagellar filament of Rhodobacter sphaeroides: pH-induced polymorphic transitions and analysis of the fliC gene

Flagellar motility in Rhodobacter sphaeroides is notably different from that in other bacteria. R. sphaeroides moves in a series of runs and stops produced by the intermittent rotation of the flagellar motor. R. sphaeroides has a single, plain filament whose conformation changes according to flagellar motor activity. Conformations adopted during swimming include coiled, helical, and apparently straight forms. This range of morphological transitions is larger than that in other bacteria, where filaments alternate between left- and right-handed helical forms. The polymorphic ability of isolated R. sphaeroides filaments was tested in vitro by varying pH and ionic strength. The isolated filaments could form open-coiled, straight, normal, or curly conformations. The range of transitions made by the R. sphaeroides filament differs from that reported for Salmonella enterica serovar Typhimurium. The sequence of the R. sphaeroides fliC gene, which encodes the flagellin protein, was determined. The gene appears to be controlled by a sigma(28)-dependent promoter. It encodes a predicted peptide of 493 amino acids. Serovar Typhimurium mutants with altered polymorphic ability usually have amino acid changes at the terminal portions of flagellin or a deletion in the central region. There are no obvious major differences in the central regions to explain the difference in polymorphic ability. In serovar Typhimurium filaments, the termini of flagellin monomers have a coiled-coil conformation. The termini of R. sphaeroides flagellin are predicted to have a lower probability of coiled coils than are those of serovar Typhimurium flagellin. This may be one reason for the differences in polymorphic ability between the two filaments.     

The effect of submergence on heart rate and oxygen consumption of swimming seals and sea lions

Respiratory, metabolic, and cardiovascular responses to swimming were examined in two species of pinniped, the harbor seal (Phoca vitulina) and the California sea lion (Zalophus californianus). 1. Harbor seals remained submerged for 82-92% of the time at swimming speeds below 1.2 m.s-1. At higher speeds, including simulated speeds above 1.4 m.s-1, the percentage of time spent submerged decreased, and was inversely related to body weight. In contrast, the percentage of time spent submerged did not change with speed for sea lions swimming from 0.5 m.s-1 to 4.0 m.s-1. 2. During swimming, harbor seals showed a distinct breathhold bradycardia and ventilatory tachycardia that were independent of swimming speed. Average heart rate was 137 beats.min-1 when swimming on the water surface and 50 beats.min-1 when submerged. A bimodal pattern of heart rate also occurred in sea lions, but was not as pronounced as in the seals. 3. The weighted average heart rate (WAHR), calculated from measured heart rate and the percentage time spent on the water surface or submerged, increased linearly with swimming speed for both species. The graded increase in heart rate with exercise load is similar to the response observed for terrestrial mammals. 4. The rate of oxygen consumption increased exponentially with swimming speed in both seals and sea lions. The minimum cost of transport calculated from these rates ranged from 2.3 to 3.6 J.m-1.kg-1, and was 2.5-4.0 times the level predicted for similarly-sized salmonids. Despite different modes of propulsion and physiological responses to swimming, these pinnipeds demonstrate similar transport costs.     

Effects of osmolality, morphology perturbations and intracellular nucleotide content during the movement of sea bass (Dicentrarchus labrax) spermatozoa.

Sea bass spermatozoa are maintained immotile in the seminal fluid, but initiate swimming for 45 s at 20 degrees C, immediately after dispersion in a hyperosmotic medium (1100 mOsm kg-1). The duration of this motile period could be extended by a reduction of the amplitude of the hyperosmotic shock. Five seconds after the initiation of motility, 94.4 +/- 1.8% of spermatozoa were motile with a swimming velocity of 141.8 +/- 1.2 microns s-1, a flagellar beat frequency of 60 Hz and a symmetric type of flagellar swimming, resulting in linear tracks. Velocity, flagellar beat frequency, percentage of motile cells and trajectory diameter decreased concomitantly throughout the swimming phase. After 30 s of motility, the flagellar beat became asymmetric, leading to circular trajectories. Ca2+ modulated the swimming pattern of demembranated spermatozoa, suggesting that the asymmetric waves produced by intact spermatozoa after 30 s of motility were induced by an accumulation of intracellular Ca2+. Moreover, increased ionic strength in the reactivation medium induced a dampening of waves in the distal portion of the flagellum and, at high values, resulted in an arrest of wave generation in demembranated spermatozoa. In non-demembranated cells, the intracellular ATP concentration fell immediately after transfer to sea water. In contrast, the AMP content increased during the same period, while the ADP content increased slightly. In addition, several morphological changes affected the mitochondria, chromatin and midpiece. These results indicate that the short swimming period of sea bass spermatozoa is controlled by energetic and cytoplasmic ionic conditions and that it is limited by osmotic stress, which induces marked changes in cell morphology.     

Identification of a chemotaxis operon with two cheY genes in Rhodobacter sphaeroides

A large chemotaxis operon was identified in Rhodobacter sphaeroides WS8-N using a probe based on the 3' terminal portion of the Rhizobium meliloti cheA gene. Two genes homologous to the enteric cheY were identified in an operon also containing cheA , cheW , and cheR homologues. The deduced protein sequences of che gene products were aligned with those from Escherichia coli and shown to be highly conserved. A mutant with an interrupted copy of cheA showed normal patterns of swimming, unlike the equivalent mutants in E. coli which are smooth swimming. Tethered cheA mutant cells showed normal responses to changes in organic acids, but increased, inverted responses to sugars. The unusual behaviour of the cheA mutant and the identification of two homologues of cheY suggests that R. sphaeroides has at least two pathways controlling motor activity. To identify functional similarity between the newly identified R. sphaeroides Che pathway and the methyl-accepting chemotaxis protein (MCP)-dependent pathway in enteric bacteria, the R. sphaeroides cheW gene was expressed in a cheW mutant strain of E. coli and found to complement, causing a partial return to a swarming phenotype. In addition, expression of the R. sphaeroides gene in wild-type E. coli resulted in the same increased tumbling and reduced swarming as seen when the native gene is over-expressed in E. coli . The identification of che homologues in R. sphaeroides and complementation by cheW suggests the presence of MCPs in an organism previously considered to use only MCP-independent sensing. The MCP-dependent pathway, appears conserved. In R. sphaeroides this pathway may mediate responses to sugars, while responses to organic acids may in involve a second system, possibly using the second CheY protein identified in this study.     

New motion analysis system for characterization of the chemosensory response kinetics of Rhodobacter sphaeroides under different growth conditions

We developed a new set of software tools that enable the speed and response kinetics of large numbers of tethered bacterial cells to be rapidly measured and analyzed. The software provides precision, accuracy, and a good signal-to-noise ratio combined with ease of data handling and processing. The software was tested on the single-cell chemosensory response kinetics of large numbers of Rhodobacter sphaeroides cells grown under either aerobic or photoheterotrophic conditions and either in chemostats or in batch cultures, allowing the effects of growth conditions on responses to be accurately measured. Aerobically and photoheterotrophically grown R. sphaeroides exhibited significantly different chemosensory response kinetics and cell-to-cell variability in their responses to 100 μM propionate. A greater proportion of the population of aerobically grown cells responded to a 100 μM step decrease in propionate; they adapted faster and showed less cell-to-cell variability than photosynthetic populations. Growth in chemostats did not significantly reduce the measured cell to cell variability but did change the adaptation kinetics for photoheterotrophically grown cells.     

Purification and characterization of the chaperonin 10 and chaperonin 60 proteins from Rhodobacter sphaeroides.

Two heat-shock proteins that show high identity with the Escherichia coli chaperonin 60 (groEL) and chaperonin 10 (groES) chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides. The proteins were purified by using sucrose density gradient centrifugation and Mono-Q anion-exchange chromatography. In the presence of 1 mM ATP, the chaperonin 10 and chaperonin 60 proteins bound to each other and comigrated as a large complex during sucrose density gradient centrifugation. The native molecular weights of each protein as determined by gel filtration chromatography were 889,200 for chaperonin 60 and 60,000 for chaperonin 10. Chaperonin 60 is comprised of monomers with a molecular weight of 61,000 and chaperonin 10 is comprised of monomers with a molecular weight of 12,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chaperonin 60 was 9.3% of the total soluble cell protein during photolithoautotrophic growth which increased to 28.5% following heat-shock treatment. When cells were grown photoheterotrophically or chemoheterotrophically, chaperonin 60 was reduced to 6.7% and 3.5%, respectively, of the total soluble protein. The N-terminal amino acid sequence of each protein was determined; chaperonin 60 of R. sphaeroides showed 72% identity to E. coli chaperonin 60 protein, and R. sphaeroides chaperonin 10 showed 45% identity with E. coli chaperonin 10. R. sphaeroides chaperonin 60 catalyzed ATP hydrolysis with a specific activity of 134 nmol min-1 mg-1 (kcat = 0.13 s-1) and was inhibited by R. sphaeroides chaperonin 10, but not E. coli chaperonin 10. The E. coli chaperonin 60 ATPase activity was inhibited by chaperonin 10 from both R. sphaeroides and E. coli.     

In Rhodobacter sphaeroides, chemotactic operon 1 regulates rotation of the flagellar system 2

Rhodobacter sphaeroides is able to assemble two different flagella, the subpolar flagellum (Fla1) and the polar flagella (Fla2). In this work, we report the swimming behavior of R. sphaeroides Fla2(+) cells lacking each of the proteins encoded by chemotactic operon 1. A model proposing how these proteins control Fla2 rotation is presented.     

Isolation and characterization of transposon Tn5 mutants of Rhodobacter sphaeroides deficient in both nitrate and chlorate reduction.

Abstract The wild-type strain Rhodobacter sphaeroides DSM 158 is a nitrate-reducing bacterium with a periplasmic nitrate reductase. Addition of chlorate to the culture medium causes a stimulation of the phototrophic growth, indicating that this strain is able to use chlorate as an ancillary oxidant. Several mutant strains of R. sphaeroides deficient in nitrate reductase activity were obtained by transposon Tn5 mutagenesis. Mutant strain NR45 exhibited high constitutive nitrate and chlorate reductase activities and phototrophic growth was also increased by the presence of chlorate. In contrast, the stimulation of growth by chlorate was not observed in mutant strains NR8 and NR13, in which transposon Tn5 insertion causes the simultaneous loss of both nitrate and chlorate reductase activities. Tn5 insertion probably does not affect molybdenum metabolism since NR8 and NR13 mutants exhibit both xanthine dehydrogenase and nitrogenase activities. These results that a single enzyme could reduce both nitrate and chlorate in R. sphaeroides DSM 158.