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Projects In Plasmid Biology (1) Mercury-resistance transposons of the Tn5053/502-family

Our interests include the epidemiology of these elements, their evolutionary relationships to each other and to Tn (or In) elements that contain related mer (HgII-resistance) and/or tni (transposition) modules, and the mechanistic basis of their unusual target-specificity and their ability (under some circumstances) to transpose randomly.

(2) Evidence of evolutionary divergence, and of conservation, in promiscuous (IncPβ) plasmids

A detailed study of several IncPβ plasmids has revealed that their conserved "backbone" has been disrupted at two locations by multiple insertions of Tn or In elements. The extant "nested transposons" are not self-mobile, but can be induced to relocate when an appropriate tnpA (transposase) in provided in trans. The varying molecular composition of the moveable elements highlights the fact that seemingly "dead" transposons can continue to contribute to bacterial diversity in unexpected ways.

Projects In Agrobacterium Biology (3) The molecular biology of EPS (extracellular polysaccharide) production

Our studies have dealt mainly with the production of curdlan by a high-yielding Agrobacterium strain like those used in commercial curdlan production. This water-insoluble EPS is produced under N-depleted conditions, is structurally simple (a linear, (1-->3)-β-glucan) and its production involves three structural genes (crdASC) whose precise roles and regulation are under investigation.

A hitherto "cryptic" and water-soluble EPS (named EPS-X) is elicited by elevated MnII levels and is co-produced with curdlan. A putative EPS-X synthesis/secretion region has been identified whose size (ca. 17 kb) suggests that EPS-X is a heterpolysaccharide. The aim of the work is to identify the essential epx genes, determine whether EPS-X is novel (i.e. its sub-unit composition, structure and pathway of synthesis) and whether there is "cross-talk" between the crd and epx production systems.

(4) The regulatory cascade leading to EPS production

We hope to determine the regulatory cascade leading to curdlan (and EPS-X) production by assessing the roles of various global regulators, namely: a two-component system (NtrBC) that senses intracellular N-status, the "alarmone" (RelA) that initiates the bacterial stringent response to environmental stress and the metalloprotease/chaperone (FtsH) that is essential for survival in stationary phase. The role of additional, positive-acting, functions that may be curdlan-specific (the CrdR regulatory protein) or EPS-X-specific (an orphan sensor kinase) are also being studied.

(5) The biological role(s) of EPS (horses for courses?)

EPS production is a complex process initiated by an interplay of various environmental factors (physical and nutritional) and cellular physiological triggers. The EPS armoury of Agrobacterium is impressive - it has the capacity to produce at least six though not simultaneously, suggesting one or more combinations of specific elicitors for each EPS. The success of Agrobacterium as a soil saprophyte may be underscored by its EPS versatility. We are studying the epidemiology of curdlan production by agrobacteria from local soils and comparing the roles of cellulose and curdlan in natural contexts (e.g. attachment to, and survival on, plant tissues; resistance to soil predators and physical stressors e.g. temperature, dessication, toxic agents).