Complete genomic nucleotide sequence and analysis of the temperate bacteriophage VWB.

The entire double-stranded DNA genome of the Streptomyces venezuelae bacteriophage VWB was sequenced and analyzed. Its size is 49,220 bp with an overall molar G + C content of 71.2 mol%. Sixty-one potential open reading frames were identified and annotated using several complementary bioinformatics tools. Clusters of functionally related putative genes were defined, supporting a refined version of the modular theory of phage evolution.

An evolutionary hot spot: the pNGR234b replicon of Rhizobium sp. strain NGR234.

Rhizobium sp. strain NGR234 has an exceptionally broad host range and is able to nodulate more than 112 genera of legumes. Since the overall organization of the NGR234 genome is strikingly similar to that of the narrow-host-range symbiont Rhizobium meliloti strain 1021 (also known as Sinorhizobium meliloti), the obvious question is why are the spectra of hosts so different? Study of the early symbiotic genes of both bacteria (carried by the SymA plasmids) did not provide obvious answers. Yet, both rhizobia also possess second megaplasmids that bear, among many other genes, those that are involved in the synthesis of extracellular polysaccharides (EPSs). EPSs are involved in fine-tuning symbiotic interactions and thus may help answer the broad- versus narrow-host-range question. Accordingly, we sequenced two fragments (total, 594 kb) that encode 575 open reading frames (ORFs). Comparisons revealed 19 conserved gene clusters with high similarity to R. meliloti, suggesting that a minimum of 28% (158 ORFs) of the genetic information may have been acquired from a common ancestor. The largest conserved cluster carried the exo and exs genes and contained 31 ORFs. In addition, nine highly conserved regions with high similarity to Agrobacterium tumefaciens C58, Bradyrhizobium japonicum USDA110, and Mesorhizobium loti strain MAFF303099, as well as two conserved clusters that are highly homologous to similar regions in the plant pathogen Erwinia carotovora, were identified. Altogether, these findings suggest that >/==" BORDER="0">40% of the pNGR234b genes are not strain specific and were probably acquired from a wide variety of other microbes. The presence of 26 ORFs coding for transposases and site-specific integrases supports this contention. Surprisingly, several genes involved in the degradation of aromatic carbon sources and genes coding for a type IV pilus were also found.

Metagenome survey of biofilms in drinking-water networks.

Most naturally occurring biofilms contain a vast majority of microorganisms which have not yet been cultured, and therefore we have little information on the genetic information content of these communities. Therefore, we initiated work to characterize the complex metagenome of model drinking water biofilms grown on rubber-coated valves by employing three different strategies. First, a sequence analysis of 650 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to the Proteobacteria: Only a small fraction of the 16S rRNA sequences were highly similar to rRNA sequences from Actinobacteria, low-G+C gram-positives and the Cytophaga-Flavobacterium-Bacteroides group. Our second strategy included a snapshot genome sequencing approach. Homology searches in public databases with 5,000 random sequence clones from a small insert library resulted in the identification of 2,200 putative protein-coding sequences, of which 1,026 could be classified into functional groups. Similarity analyses indicated that significant fractions of the genes and proteins identified were highly similar to known proteins observed in the genera Rhizobium, Pseudomonas, and Escherichia: Finally, we report 144 kb of DNA sequence information from four selected cosmid clones, of which two formed a 75-kb overlapping contig. The majority of the proteins identified by whole-cosmid sequencing probably originated from microbes closely related to the alpha-, beta-, and gamma-Proteobacteria: The sequence information was used to set up a database containing the phylogenetic and genomic information on this model microbial community. Concerning the potential health risk of the microbial community studied, no DNA or protein sequences directly linked to pathogenic traits were identified.

Prospecting for novel biocatalysts in a soil metagenome.

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an alpha-amylase (amyA), a 1,4-alpha-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.

The crystal structure of Thermotoga maritima maltosyltransferase and its implications for the molecular basis of the novel transfer specificity.

Maltosyltransferase (MTase) from the hyperthermophile Thermotoga maritima represents a novel maltodextrin glycosyltransferase acting on starch and malto-oligosaccharides. It catalyzes the transfer of maltosyl units from alpha-1,4-linked glucans or malto-oligosaccharides to other alpha-1,4-linked glucans, malto-oligosaccharides or glucose. It belongs to the glycoside hydrolase family 13, which represents a large group of (beta/alpha)(8) barrel proteins sharing a similar active site structure. The crystal structures of MTase and its complex with maltose have been determined at 2.4 A and 2.1 A resolution, respectively. MTase is a homodimer, each subunit of which consists of four domains, two of which are structurally homologous to those of other family 13 enzymes. The catalytic core domain has the (beta/alpha)(8) barrel fold with the active-site cleft formed at the C-terminal end of the barrel. Substrate binding experiments have led to the location of two distinct maltose-binding sites; one lies in the active-site cleft, covering subsites -2 and -1; the other is located in a pocket adjacent to the active-site cleft. The structure of MTase, together with the conservation of active-site residues among family 13 glycoside hydrolases, are consistent with a common double-displacement catalytic mechanism for this enzyme. Analysis of maltose binding in the active site reveals that the transfer of dextrinyl residues longer than a maltosyl unit is prevented by termination of the active-site cleft after the -2 subsite by the side-chain of Lys151 and the stretch of residues 314-317, providing an explanation for the strict transfer specificity of MTase.

Crystallization and preliminary X-ray crystallographic studies on 4-alpha-glucanotransferase from Thermotoga maritima.

Thermotoga maritima 4-alpha-glucanotransferase (GTase), a 52 kDa molecular-weight amylolytic enzyme, has been crystallized by the hanging-drop vapour-diffusion method using PEG monomethylether 5000 as a precipitating agent. A complete data set has been collected to 2.6 A resolution using cryocooling conditions and synchrotron radiation. The crystals belong to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 92.6, b = 180.3, c = 199.2 A.

Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent alpha-glucosidase.

The gene for the alpha-glucosidase AglA of the hyperthermophilic bacterium Thermotoga maritima MSB8, which was identified by phenotypic screening of a T. maritima gene library, is located within a cluster of genes involved in the hydrolysis of starch and maltodextrins and the uptake of maltooligosaccharides. According to its primary structure as deduced from the nucleotide sequence of the gene, AglA belongs to family 4 of glycosyl hydrolases. The enzyme was recombinantly expressed in Escherichia coli, purified, and characterized. The T. maritima alpha-glucosidase has the unusual property of requiring NAD+ and Mn2+ for activity. Co2+ and Ni2+ also activated AglA, albeit less efficiently than Mn2+. T. maritima AglA represents the first example of a maltodextrin-degrading alpha-glucosidase with NAD+ and Mn2+ requirement. In addition, AglA activity depended on reducing conditions. This third requirement was met by the addition of dithiothreitol (DTT) or beta-mercaptoethanol to the assay. Using gel permeation chromatography, T. maritima AglA behaved as a dimer (two identical 55-kDa subunits), irrespective of metal depletion or metal addition, and irrespective of the presence or absence of NAD+ or DTT. The enzyme hydrolyzes maltose and other small maltooligosaccharides but is inactive against the polymeric substrate starch. AglA is not specific with respect to the configuration at the C-4 position of its substrates because glycosidic derivatives of D-galactose are also hydrolyzed. In the presence of all cofactors, maximum activity was recorded at pH 7.5 and 90 degrees C (4-min assay). AglA is the most thermoactive and the most thermostable member of glycosyl hydrolase family 4. When incubated at 50 degrees C and 70 degrees C, the recombinant enzyme suffered partial inactivation during the first hours of incubation, but thereafter the residual activity did not drop below about 50% and 20% of the initial value, respectively, within a period of 48 h.

Crystallization and preliminary X-ray crystallographic studies on maltosyltransferase from Thermotoga maritima.

Thermotoga maritima maltosyltransferase (MTase) is a 73.7 kDa molecular weight amylolytic enzyme which catalyzes the transfer of maltosyl units from maltodextrins or starch to suitable acceptors. Crystals of recombinant MTase have been obtained by the hanging-drop vapour-diffusion method using ammonium phosphate as a precipitating agent. The crystals belong to space group P4(1)22 or its enantiomorph P4(3)22, with unit-cell parameters a = b = 148.7, c = 106.7 A. The asymmetric unit appears to contain one subunit, corresponding to a very low packing density of 4.0 A(3) Da(-1). The crystals diffract X-rays to at least 2.4 A resolution on a synchrotron-radiation source.

Locomotor strategy for pedaling: muscle groups and biomechanical functions.

A group of coexcited muscles alternating with another group is a common element of motor control, including locomotor pattern generation. This study used computer simulation to investigate human pedaling with each muscle assigned at times to a group. Simulations were generated by applying patterns of muscle excitations to a musculoskeletal model that includes the dynamic properties of the muscles, the limb segments, and the crank load. Raasch et al. showed that electromyograms, pedal reaction forces, and limb and crank kinematics recorded during maximum-speed start-up pedaling could be replicated with two signals controlling the excitation of four muscle groups (1 group alternating with another to form a pair). Here a four-muscle-group control also is shown to replicate steady pedaling. However, simulations show that three signals controlling six muscle groups (i.e., 3 pairs) is much more biomechanically robust, such that a wide variety of forward and backward pedaling tasks can be executed well. We found the biomechanical functions necessary for pedaling, and how these functions can be executed by the muscle groups. Specifically, the phasing of two pairs with respect to limb extension and flexion and the transitions between extension and flexion do not change with pedaling direction. One pair of groups (uniarticular hip and knee extensors alternating with their anatomic antagonists) generates the energy required for limb and crank propulsion during limb extension and flexion, respectively. In the second pair, the ankle plantarflexors transfer the energy from the limb inertia to the crank during the latter part of limb extension and the subsequent limb extension-to-flexion transition. The dorsiflexors alternate with the plantarflexors. The phasing of the third pair (the biarticular thigh muscles) reverses with pedaling direction. In forward pedaling, the hamstring is excited during the extension-to-flexion transition and in backward pedaling during the opposite transition. In both cases hamstrings propel the crank posteriorly through the transition. Rectus femoris alternates with hamstrings and propels the crank anteriorly through the transitions. With three control signals, one for each pair of groups, different cadences (or power outputs) can be achieved by adjusting the overall excitatory drive to the pattern generating elements, and different pedaling goals (e.g., smooth, or energy-efficient pedaling; 1- or 2-legged pedaling) by adjusting the relative excitation levels among the muscle groups. These six muscle groups are suggested to be elements of a general strategy for pedaling control, which may be generally applicable to other human locomotor tasks.

Sensorimotor state of the contralateral leg affects ipsilateral muscle coordination of pedaling.

The objective of this study was to determine if independent central pattern generating elements controlling the legs in bipedal and unipedal locomotion is a viable theory for locomotor propulsion in humans. Coordinative coupling of the limbs could then be accomplished through mechanical interactions and ipsilateral feedback control rather than through central interlimb neural pathways. Pedaling was chosen as the locomotor task to study because interlimb mechanics can be significantly altered, as pedaling can be executed with the use of either one leg or two legs (cf. walking) and because the load on the limb can be well-controlled. Subjects pedaled a modified bicycle ergometer in a two-legged (bilateral) and a one-legged (unilateral) pedaling condition. The loading on the leg during unilateral pedaling was designed to be identical to the loading experienced by the leg during bilateral pedaling. This loading was achieved by having a trained human "motor" pedal along with the subject and exert on the opposite crank the torque that the subject's contralateral leg generated in bilateral pedaling. The human "motor" was successful at reproducing each subject's one-leg crank torque. The shape of the motor's torque trajectory was similar to that of subjects, and the amount of work done during extension and flexion was not significantly different. Thus the same muscle coordination pattern would allow subjects to pedal successfully in both the bilateral and unilateral conditions, and the afferent signals from the pedaling leg could be the same for both conditions. Although the overall work done by each leg did not change, an 86% decrease in retarding (negative) crank torque during limb flexion was measured in all 11 subjects during the unilateral condition. This corresponded to an increase in integrated electromyography of tibialis anterior (70%), rectus femoris (43%), and biceps femoris (59%) during flexion. Even given visual torque feedback in the unilateral condition, subjects still showed a 33% decrease in negative torque during flexion. These results are consistent with the existence of an inhibitory pathway from elements controlling extension onto contralateral flexion elements, with the pathway operating during two-legged pedaling but not during one-legged pedaling, in which case flexor activity increases. However, this centrally mediated coupling can be overcome with practice, as the human "motor" was able to effectively match the bilateral crank torque after a longer practice regimen. We conclude that the sensorimotor control of a unipedal task is affected by interlimb neural pathways. Thus a task performed unilaterally is not performed with the same muscle coordination utilized in a bipedal condition, even if such coordination would be equally effective in the execution of the unilateral task.

Muscle coordination of maximum-speed pedaling.

A simulation based on a forward dynamical musculoskeletal model was computed from an optimal control algorithm to understand uni- and bi-articular muscle coordination of maximum-speed startup pedaling. The muscle excitations, pedal reaction forces, and crank and pedal kinematics of the simulation agreed with measurements from subjects. Over the crank cycle, uniarticular hip and knee extensor muscles provide 55% of the propulsive energy, even though 27% of the amount they produce in the downstroke is absorbed in the upstroke. Only 44% of the energy produced by these muscles during downstroke is delivered to the crank directly. The other 56% is delivered to the limb segments, and then transferred to the crank by the ankle plantarflexors. The plantarflexors, especially soleus, also prevent knee hyperextension, by slowing the knee extension being produced during downstroke by the other muscles, including hamstrings. Hamstrings and rectus femoris make smooth pedaling possible by propelling the crank through the stroke transitions. Other simulations showed that pedaling can be performed well by partitioning all the muscles in a leg into two pairs of phase-controlled alternating functional groups, with each group also alternating with its contralateral counterpart. In this scheme, the uniarticular hip/knee extensor muscles (one group) are excited during downstroke, and the uniarticular hip/knee flexor muscles (the alternating group) during upstroke. The ankle dorsiflexor and rectus femoris muscles (one group of the other pair) are excited near the transition from upstroke to downstroke, and the ankle plantarflexors and hamstrings muscles (the alternating group) during the downstroke to upstroke transition. We conclude that these alternating functional muscle groups might represent a centrally generated primitive for not only pedaling but also other locomotor tasks as well.