Characterization of mycelial morphology is important for physiological and engineering studies of filamentous fermentations, and in the design and operation of such fermentations. Image analysis has been developed as a method for this characterization, and has been shown to be faster and generally more accurate than previous methods. A fully automatic system has been developed, in which speed is gained, but with loss of accuracy in some cases. The method has been tested on Streptomyces clavuligerus and Penicillium chrysogenum P1 batch fermentations. It has also been tested on a fed-batch Penicillium chrysogenum P2 fermentation, in which the medium contained solid ingredients. Fully automatic image analysis for morphological characterization of filamentous microorganisms is an important development which will make practical many engineering and physiological studies of such fermentations that have so far not been completely satisfactory.

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 overexpressed 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.(ABSTRACT TRUNCATED AT 250 WORDS)

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 swvimming 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 chemoefectors 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 frequenc 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 mi. 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.Rhodobacter sphaeroides has a single subpolar flagellum which it rotates unidirectionally. The rotation of the flagellum stops periodically for intervals of between a few seconds and many minutes. Brownian motion reorientates the cell during stopped periods, enabling changes in swimming direction (2). Although R sphaeroides lacks methyl-accepting chemotaxis proteins (MCPs) and the phosphotransferase system-dependent chemotaxis systems found in enteric bacteria (3,20,28), it is able to respond chemotactically to a wide range of attractants (21). All compounds shown to be chemoattractants for R sphaeroides are metabolites, and at least limited metabolism seems to be required for the chemotactic response (21,25). Chemotaxis in enteric bacteria and many other species, on the other hand, relies on the extracytoplasmic detection of chemoeffectors by specific MCPs which signal changes in occupancy to the f...

A methodology for the estimation of biomass for the penicillin fermentation using image analysis is presented. Two regions of hyphae are defined to describe the growth of mycelia during fermentation: (1) the cytoplasmic region, and (2) the degenerated region including large vacuoles. The volume occupied by each of these regions in a fixed volume of sample is estimated from area measurements using image analysis. Areas are converted to volumes by treating the hyphae as solid cylinders with the hyphal diameter as the cylinder diameter. The volumes of the cytoplasmic and degenerated regions are converted into dry weight estimations using hyphal density values available from the literature. The image analysis technique is able to estimate biomass even in the presence of nondissolved solids of a concentration of up to 30 gL(-1). It is shown to estimate successfully concentrations of mycelia from 0.03 to 38 gL(-1). Although the technique has been developed for the penicillin fermentation, it should be applicable to other (nonpellected) fungal fermentations.

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.

The behavioural response of Rhodobacter sphaemides to temporal changes in the concentration of chemoeffectors, and to stimuli affecting electron transport, was analysed using tethered cells. Populations of photosynthetically grown tethered cells of R. sphaemides showed a transient response, a stop followed by adaptation, to a stepwise reduction in the concentration of chemoattractants (such as organic acids or sugars) and terminal electron acceptors. A step-down response was also measured in free swimming ceHs to a reduction in light intensity. As this response appears to apply to all effectors this suggests that there is a sensory pathway in anaerobically grown R. sphaemides which responds primarily to a reduction in a stimulus. R. sphaemides therefore responds when moving down a concentration gradient. This is the inverse of the classical Escherichie coli-2almonella typhimurium model of bacterial sensory behaviour in which bacteria respond primarily when there is an increase in an attractant concentration, i.e. when moving up a gradient. R. sphaemides does show a chemokinetic response to an increase in concentration of a limited number of compounds but this response is sustained and accompanied by an increase in the rate of flagellar rotation and therefore not simply equivalent to the transient increase in smooth swimming measured in E. d i on addition of an attractant.

The temporal and spatial behavior of a number of mutants of the photosynthetic, facultative anaerobe Rhodobacter sphaeroides to both step changes and to gradients of oxygen was analyzed. Wild-type cells, grown under a range of conditions, showed microaerophilic behavior, accumulating in a 1.3-mm band about 1.3 mm from the meniscus of capillaries. Evidence suggests this is the result of two signaling pathways. The strength of any response depended on the growth and incubation conditions. Deletion of either the complete chemosensory operons 1 and 2 plus the response regulator genes cheY 4 and cheY 5 or cheA 2 alone led to the loss of all aerotactic responses, although the cells still swam normally. The Prr system of R. sphaeroides responds to electron flow through the alternative high-affinity cytochrome oxidase, cbb 3 , controlling expression of a wide range of metabolic pathways. Mutants with deletions of either the complete Prr operon or the histidine kinase, PrrB, accumulated up to the meniscus but still formed a thick band 1.3 mm from the aerobic interface. This indicates that the negative aerotactic response to high oxygen levels depends on PrrB, but the mutant cells still retain the positive response. Tethered PrrB ؊ cells also showed no response to a step-down in oxygen concentration, although those with deletions of the whole operon showed some response. In gradients of oxygen where the concentration was reduced at 0.4 M/s, tethered wild-type cells showed two different phases of response, with an increase in stopping frequency when the oxygen concentration fell from 80 to 50% dissolved oxygen and a decrease in stopping at 50 to 20% dissolved oxygen, with cells returning to their normal stopping frequency in 0% oxygen. PrrB and CheA 2 mutants showed no response, while PrrCBA mutants still showed some response.Tactic responses are a common feature of motile bacteria. A sensory system converts environmental signals into a change in the direction of rotation of the flagellar motor, resulting in the accumulation of microbial populations in microenvironments optimal for growth (1).Taxis towards oxygen (aerotaxis) has been identified in several bacterial species (45). It allows bacteria to migrate to concentrations of dissolved oxygen ideal for their current metabolism. In many species investigated, an active respiratory chain and Che proteins, such as CheA, CheW, and CheY (20,21,35), are essential for responses. Recent reports have identified a methyl-accepting chemotaxis protein (MCP)-like oxygen receptor, Aer, in Escherichia coli (2, 31) and Pseudomonas putida (26), whereas myoglobin-like aerotaxis transducers have been identified in Halobacterium salinarum and Bacillus subtilis (16) and putative oxygen transducers with a c-type heme-sensing domain have been identified in other species (4, 8), suggesting that several independent systems have evolved to control tactic behavior in response to changes in oxygen level.Rhodobacter sphaeroides is a facultative non-sulfur-photosynthetic bacterium belonging to the ␣-...

The rotation rate of the unidirectional stop/start motor of Rhodobacter sphaeroides was investigated using computerised motion analysis of tethered cells. The R. sphaeroides motor was found to have a variable rotation rate compared to the virtually constant-speed motor of wild-type and CheR mutant (smooth swimming) Escherichia coli. In addition, the dynamics of the R. sphaeroides motor during stopping was analysed with no consistent correlation behaviour. The motor could go from full rotation to stop, or stop to full rotation within one video frame, i.e. 0.02 s, but it could also slow down into a stop or restart slowly, taking up to 0.25 s. The R. sphaeroides motor under chemokinetic stimulation was also analysed and was found to show increased torque generation and reduced variation in rotation rate.© 1997 Federation of European Biochemical Societies.