Insights into the Geobacillus stearothermophilus species based on phylogenomic principles.

BACKGROUND: The genus Geobacillus comprises bacteria that are Gram positive, thermophilic spore-formers, which are found in a variety of environments from hot-springs, cool soils, to food manufacturing plants, including dairy manufacturing plants. Despite considerable interest in the use of Geobacillus spp. for biotechnological applications, the taxonomy of this genus is unclear, in part because of differences in DNA-DNA hybridization (DDH) similarity values between studies. In addition, it is also difficult to use phenotypic characteristics to define a bacterial species. For example, G. stearothermophilus was traditionally defined as a species that does not utilise lactose, but the ability of dairy strains of G. stearothermophilus to use lactose has now been well established. RESULTS: This study compared the genome sequences of 63 Geobacillus isolates and showed that based on two different genomic approaches (core genome comparisons and average nucleotide identity) the Geobacillus genus could be divided into sixteen taxa for those Geobacillus strains that have genome sequences available thus far. In addition, using Geobacillus stearothermophilus as an example, we show that inclusion of the accessory genome, as well as phenotypic characteristics, is not suitable for defining this species. For example, this is the first study to provide evidence of dairy adaptation in G. stearothermophilus - a phenotypic feature not typically considered standard in this species - by identifying the presence of a putative lac operon in four dairy strains. CONCLUSIONS: The traditional polyphasic approach of combining both genotypic and phenotypic characteristics to define a bacterial species could not be used for G. stearothermophilus where many phenotypic characteristics vary within this taxon. Further evidence of this discordant use of phenotypic traits was provided by analysis of the accessory genome, where the dairy strains contained a putative lac operon. Based on the findings from this study, we recommend that novel bacterial species should be defined using a core genome approach.

Characterization of thermophilic bacilli from a milk powder processing plant.

AIMS: To determine whether strains of Geobacillus stearothermophilus isolated from a milk powder manufacturing plant were different in their ability to form biofilms and produce spores. In addition, this study evaluated whether there were other physiological characteristics that could differentiate these strains. METHODS AND RESULTS: Ten G. stearothermophilus strains and one Anoxybacillus species were isolated from a milk powder manufacturing plant. A microtitre plate assay was used to show that these strains differed in their abilities to form biofilms and produce spores. Scanning electron microscopy showed differences in the biofilm morphologies of three of the G. stearothermophilus strains. Biochemical profiling, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and fatty acid profiling further showed that they had distinct characteristics. CONCLUSIONS: These G. stearothermophilus strains, isolated from the same environment, showed differences in their ability to form biofilms and produce endospores. Based on the multiple characterization methods used in this study, these strains of G. stearothermophilus isolated from one manufacturing plant are diverse. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences in the ability of G. stearothermophilus to form biofilms and produce spores may influence the cleaning method used to control the growth of thermophilic bacilli in a dairy processing environment.

Flexibility within myosin heads revealed by negative stain and single-particle analysis.

Electron microscopy of negatively stained myosin has previously revealed three discrete regions within the heads of the molecule. However, despite a probable resolution of approximately 2 nm, it is difficult to discern directly consistent details within these regions. This is due to variability in both head conformation and in staining. In this study, we applied single-particle image processing and classified heads into homogeneous groups. The improved signal-to-noise ratio after averaging these groups reveals substantially improved detail. The image averages were compared to a model simulating negative staining of the atomic structure of subfragment-1 (S1). This shows that the three head regions correspond to the motor domain and the essential and regulatory light chains. The image averages were very similar to particular views of the S1 model. They also revealed considerable flexibility between the motor and regulatory domains, despite the molecules having been prepared in the absence of nucleotide. This flexibility probably results from rotation of the regulatory domain about the motor domain, where the relative movement of the regulatory light chain is up to 12 nm, and is most clearly illustrated in animated sequences (available at http://www.leeds.ac.uk/chb/muscle/myosinhead.htm l). The sharply curved conformation of the atomic model of S1 is seen only rarely in our data, with straighter heads being more typical.

The formation of spores in biofilms of Anoxybacillus flavithermus.

AIMS: To examine the rate and the extent of spore formation in Anoxybacillus flavithermus biofilms and to test the effect of one key variable - temperature - on spore formation. METHODS AND RESULTS: A continuous flow laboratory reactor was used to grow biofilms of the typical dairy thermophile A. flavithermus (strain CM) in skim milk. The reactor was inoculated with either a washed culture or a spore suspension of A. flavithermus CM, and was run over an 8.5 h period at three different temperatures of 48, 55 and 60 degrees C. Change in impedance was used to determine the cell numbers in the milk and on the surface of the stainless steel reactor tubes. The biofilm developed at all three temperatures within 6-8 h. Spores formed at 55 and 60 degrees C and amounted to approx. 10-50% of the biofilm. No spores formed at 48 degrees C. CONCLUSIONS: The results suggest that both biofilm formation and spore formation of A. flavithermus can occur very rapidly and simultaneously. In addition, temperature variation has a considerable effect on the formation of spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This information will provide direction for developing improved ways in which to manipulate conditions in milk powder manufacturing plants to control biofilms and spores of A. flavithermus.

Rigor and relaxed outer dynein arms in replicas of cryofixed motile flagella.

A method of image classification based on multivariate statistics has been developed and applied to freeze-etch images of outer dynein arms (ODAs) from cockerel sperm flagella. Demembranated flagella were cryofixed in three different nucleotide states: rigor (i.e. no added ATP); relaxed (i.e. 1 mM ATP plus vanadate); and active (i.e. 1 mM ATP). Freeze-etch replica fragments from them were coded to conceal their identities and sampled for flagella. From these a total of 6048 individual ODA images were successfully windowed and aligned, covering an angular range of 66 degrees. Each nucleotide condition produced a statistically significant ODA morphology. The relaxed and active morphologies differed only in the angulation of their heads, with the relaxed ODA favouring a more tilted position. The rigor morphology was more distinct and showed a conformational change involving a 12 nm distal shift relative to the A-tubule and highly variable heads and B-links, suggesting an ability to develop tension. Outer arms were classified by discriminant analysis as being either rigor-like, relaxed-like or active-like, and 80% of all ODAs were correctly classified. The misclassification of the remaining 20% indicated morphological heterogeneity in some of the groups. From the rigor group, 8.8% of the ODAs were misclassified as relaxed-like or active-like (i.e. non-rigor-like). It is speculated that this is a consequence of mechanochemical interactions between the B-tubules and the rigor ODAs and/or contaminating ATP remaining after demembranation. Active flagella were found to show all three morphologies, with 5.4% in the rigor conformation and 18.6% in the relaxed conformation. The finding of rigor-like and relaxed-like ODAs in flagella exposed to ATP is discussed in relation to the cross-bridge cycle.

A new model for the surface arrangement of myosin molecules in tarantula thick filaments.

Three-dimensional reconstructions of the negatively stained thick filaments of tarantula muscle with a resolution of 50 A have previously suggested that the helical tracks of myosin heads are zigzagged, short diagonal ridges being connected by nearly axial links. However, surface views of lower contour levels reveal an additional J-shaped feature approximately the size and shape of a myosin head. We have modelled the surface array of myosin heads on the filaments using as a building block a model of a two-headed regulated myosin molecule in which the regulatory light chains of the two heads together form a compact head-tail junction. Four parameters defining the radius, orientation and rotation of each myosin molecule were varied. In addition, the heads were allowed independently to bend in a plane perpendicular to the coiled-coil tail at three sites, and to tilt with respect to the tail and to twist at one of these sites. After low-pass filtering, models were aligned with the reconstruction, scored by cross-correlation and refined by simulated annealing. Comparison of the geometry of the reconstruction and the distance between domains in the myosin molecule narrowed the choice of models to two main classes. A good match to the reconstruction was obtained with a model in which each ridge is formed from the motor domain of a head pointing to the bare zone together with the head-tail junction of a neighbouring molecule. The heads pointing to the Z-disc intermittently occupy the J-position. Each motor domain interacts with the essential and regulatory light chains of the neighbouring heads. A near-radial spoke in the reconstruction connecting the backbone to one end of the ridge can be identified as the start of the coiled-coil tail.

Architecture of the outer arm dynein ATPase in an avian sperm flagellum, with further evidence for the B-link.

Demembranated sperm flagella from Gallus domesticus have been prepared by the rapid-freeze, deep-etch, rotary replica technique in order to study the three-dimensional morphology of the outer dynein arm (ODA)-ATPase complex. In general, the ODAs resemble most closely those from the sea urchin Strongylocentrotus described by W. S. Sale et al. In the 'rigor' condition, the ODA consists of a major subunit (the head), from which a slender link extends to the adjacent B-tubule (the B-link). A smaller, intermediate subunit lies adjacent to the head, distally, and a further extension of the complex, the minor subunit, continues distally beneath the head domain of the next arm complex, where it attaches to the A-tubule. In the presence of ATP and vanadate ('relaxed' condition), the attachment point of the B-link to the head is shifted to a more proximal position, and the minor subunit is no longer visible. This is interpreted as resulting from a rotation of the head. These features are demonstrated stereoscopically, and from several viewpoints. Image enhancement has been used to clarify and define the repetitive features of the dynein arrays. In addition, some of the axonemes have been imaged from highly contrasted 20 nm thin sections; the detection of B-links in such sections means that these slender structures cannot be considered artefacts of the rapid-freeze, deep-etch protocol.

The inner dynein arm complex: compatible images from freeze-etch and thin section methods of microscopy.

A study has been made of the inner dynein arm complex in the sperm flagellum of Gallus domesticus. It has been found that images of the complex made from highly contrasted, approx. 20 nm, sections are very largely compatible with images made from replicas of rapidly cryofixed material. This suggests that neither technique seriously distorts the native state. From both types of image, we conclude that the most proximal group of inner dynein arm heads (IDA 1) is related to spoke S1 and consists of 3 heads with fine connections to the B-tubule. IDA 2 consists of 2 such heads and is related to spoke S2. IDA 3 is apparently single headed and lies close to the A-attachment of spoke S3. This arrangement of IDAs repeats every 96 nm. Between the IDAs and outer dynein arms (ODAs), lying close to IDAs 2 and 3, is a strap-like linkage to the next B-tubule; it is argued that this represents the circumferential link, nexin. In sections, but not consistently in replicas, IDA2 lies closer to the plane of the nexin link than does IDA 1. In the presence of ATP and vanadate, little change is seen in the IDA complex except for an ill-defined alteration to IDA 1. It is speculated that the apparently smaller size of the inner arm heads (compared with the ODA major subunit) may have functional implications in relation to maximal sliding velocity.

The structure of dynein-c by negative stain electron microscopy.

Dynein ATPases contain six concatenated AAA modules within the motor region of their heavy chains. Additional regions of sequence are required to form a functional ATPase, which a previous study suggested forms seven or eight subdomains arranged in either a ring or hollow sphere. A more recent homology model of the six AAA modules suggests that these form a ring. Therefore both the number and arrangement of subdomains remain uncertain. We show two-dimensional projection images of dynein-c in negative stain which reveal new details of its structure. Initial electron cryomicroscopy shows a similar overall morphology. The molecule consists of three domains: stem, head, and stalk. In the absence of nucleotide the head has seven lobes of density forming an asymmetric ring. An eighth lobe protrudes from one side of this heptameric ring and appears to join the elongated cargo-binding stem. The proximal stem is flexible, as is the stalk, suggesting that they act as compliant elements within the motor. A new analysis of pre- and post-power stroke conformations shows the combined effect of their flexibility on the spatial distribution of the microtubule-binding domain and therefore the potential range of power stroke sizes. We present and compare two alternative models of the structure of dynein.

Reinnervation of sweat glands in the rat hind paw following peripheral nerve injury.

Electrophysiological experiments have been carried out to investigate the time course and extent of sweat gland reinnervation in the rat hind paw. The first evidence of functional reinnervation after nerve transection was obtained at 12 weeks, when the extent of innervation was 20% of that measured in control animals. By 20 weeks, reinnervation had reached almost 50% of control values but then there was no further improvement up to 52 weeks. These results are comparable to those for skin reinnervation by polymodal nociceptor afferents.

Three-dimensional structure of type IV collagen in the mammalian lens capsule.

The anterior lens capsule provides a thick, easily handled model system for the study of the organization of type IV collagen, the main component of basement membranes. We have used the technique of rapid freezing, deep-etch, and rotary replication to study the three-dimensional organization of the collagen skeleton in mammalian lens capsule after a variety of extraction procedures. In all cases the collagen appeared as a densely packed three-dimensional branching network of fine microfibrils. The organization of the microfibrils appears to show some regularity, with branch points approximately 40 nm apart. Most junctions are three-way and the network forms predominantly five-sided figures. This closely resembles the collagenous network described by Yurchenco and Ruben (1987, 1988) in human amniotic basement membrane and EHS tumor matrix, but extends their findings to another system for which X-ray diffraction data are available. The three-dimensional network is discussed in terms of molecular packing of type IV collagen in light of the information available from the diffraction data.

Two-headed binding of a processive myosin to F-actin.

Myosins are motor proteins in cells. They move along actin by changing shape after making stereospecific interactions with the actin subunits. As these are arranged helically, a succession of steps will follow a helical path. However, if the myosin heads are long enough to span the actin helical repeat (approximately 36 nm), linear motion is possible. Muscle myosin (myosin II) heads are about 16 nm long, which is insufficient to span the repeat. Myosin V, however, has heads of about 31 nm that could span 36 nm and thus allow single two-headed molecules to transport cargo by walking straight. Here we use electron microscopy to show that while working, myosin V spans the helical repeat. The heads are mostly 13 actin subunits apart, with values of 11 or 15 also found. Typically the structure is polar and one head is curved, the other straighter. Single particle processing reveals the polarity of the underlying actin filament, showing that the curved head is the leading one. The shape of the leading head may correspond to the beginning of the working stroke of the motor. We also observe molecules attached by one head in this conformation.