Coordination of outer membrane constriction with septation is critical to faithful division in Gram-negative bacteria and vital to the barrier function of the membrane. This coordination requires the recruitment of the peptidoglycan-binding outer-membrane lipoprotein Pal at division sites by the Tol system. Here, we show that Pal accumulation at Escherichia coli division sites is a consequence of three key functions of the Tol system. First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly due to their binding of the cell wall. Second, Tol actively captures mobilised Pal molecules and deposits them at the division septum. Third, the active capture mechanism is analogous to that used by the inner membrane protein TonB to dislodge the plug domains of outer membrane TonB-dependent nutrient transporters. We conclude that outer membrane constriction is coordinated with cell division by active mobilisation-and-capture of Pal at division septa by the Tol system.

Background: pH sensitivity differences between skeletal and cardiac muscle originate from distinct troponin I isoforms. Results: Histidine 130 in skeletal troponin I, absent in the cardiac isoform, makes an electrostatic interaction with cardiac troponin C at low pH. Conclusion: This interaction compensates for decreased calcium affinity under acidic conditions. Significance: This understanding may aid in the development of therapies that reverse the negative inotropic effects of acidosis.

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A survey of the literature based on the accumulated data of Block and Boiling (1951) indicates that knowledge of the amino-acid composition of the proteins of legume seeds is limited. There is an almost complete lack of comprehensive data for the seeds of legumes of particular importance to Australian agriculture, such as lupins, vetches, peas and subterranean clover. Investigations of the digestibility and biological value of the protein of some of these seeds, using nitrogen balance methods, have been reported from this laboratory for the growth of sheep (Williams and Moir, 1951) and for the growth of the rat (Smythe, 1950) but they were not supported by any extensive data on their amino-acid composition. The arginine content of these seed proteins was, however, reported by the author (Holmes, 1951) and the cyst(e)ine and methionine contents of some of them by Johanson (1948).In recent years considerable attention has been devoted to the sulphurcontaining amino-acids in seed proteins (Lugg, 1945 and 194C; Johanson and Lugg, 1946). As a result it has beeii establislied that legume seeds and their bulk proteins are of very low or low methionine content and their cyst(e)ine values range from very low to moderately high. Numerous feeding trials indicate that the methionine contents of legume seeds may constitute a first limiting factor in nutrition, at least in the rat and the chick.' On the other hand, in spite of the not unexpected emphasis placed on the sulphur-containing amino-acids in wool growth (Marston, 1935) no correlation could be found by Johanson (loc. cit.) between cyst(e)ine + methionine fed and wool growth in the series of feeding trials carried out by Stewart and Moir (1947). In these trials marked differences were found between different protein sources, including several legume seeds, in promoting wool growth in Merino sheep when these proteins were fed at the same overall level of nitrogen intake and constituted almost the whole of the nitrogen of otherwise similar rations. The possibility of other essential amino-acids limiting the wool-growing capacity of these proteins, particularly those occurring in wool keratin in high proportions,, namely arginine, leucine and threonine, could not be excluded. The present study of the amino-acid composition of certain legume seed proteins and of linseed protein.

It has been suggested (Lugg and Weller, 1944) that for the purpose of animal nutrition studies, comprehensive analyses of pasture proteins might be confined to a few species belonging to the most important families of pasture plants. In the case of special proteins such as the seed proteins, however, no such relatively uniform amino acid composition exists. Partial amino acid analyses of seed proteins must then, of necessity, be made with the individual types of seedis themselves for use in animal nutrition studies, as even closely related seed proteins exhibit very varied amino acid compositions.Arginine contents of certain seed proteins have been determined, the analyses being made by Vickery's (1940) modified flavianic acid method and based on "whole" protein preparations of the seeds. The seeds selected for analysis are those commorlly used as a protein source for sheep during the summer months. It is intended to extend the partial amino acid analyses of these seeds, particularly for those amino acids listed as being nutritionally essential in the diet (Rose, 1938) and, in a subsequent paper, to discuss the nutritional significance of the results in relation to the wool growth of Merino sheep. EXPERIMENTAL. Preparation of materials.The seeds selected for analysis were Lupin (Lupinus pilo.ms W.A. blue); Pea (Fisum sativum, var. White Brunswick); Vetch (Vicia satimim) ; Subterranean Clover (Trifoliiim .subterraneum) and a commercial product of Linseed (Linum usitatissimum).

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25Coordination of outer membrane constriction with septation is critical to faithful division 26 in Gram-negative bacteria and vital to the barrier function of the membrane. Recent 27 studies suggest this coordination is through the active accumulation of the 28 peptidoglycan-binding outer membrane lipoprotein Pal at division sites by the Tol 29 system, but the mechanism is unknown. Here, we show that Pal accumulation at 30Escherichia coli division sites is a consequence of three key functions of the Tol system. 31First, Tol mobilises Pal molecules in dividing cells, which otherwise diffuse very slowly 32 due to their binding of the cell wall. Second, Tol actively captures mobilised Pal 33 molecules and deposits them at the division septum. Third, the active capture 34 mechanism is analogous to that used by the inner membrane protein TonB to dislodge 35 the plug domains of outer membrane TonB-dependent nutrient transporters. We 36 conclude that outer membrane constriction is coordinated with cell division by active 37 mobilisation-and-capture of Pal at division septa by the Tol system. 38 39 Word count, 160 40 41 42 43 44 45 46 47 across the inner membrane 7-9 . Recently, Petiti et al have suggested that the 60multiprotein Tol system (also known as Tol-Pal) constricts the OM by populating the 61 division septum with Pal 10 . However, no mechanism has been proposed. In the present 62 work, through a combination of in vivo imaging, deletion analysis and mutagenesis, 63 structure determination, biophysical measurements, mathematical modelling and 64 molecular dynamics simulations, we demonstrate that the PMF is exploited by the Tol 65 system to both mobilise Pal in the OM of dividing cells and to then capture these 66 mobilised molecules at division sites. Mobilisation-and-capture circumvents Pal's 67 intrinsically low mobility in the OM and results in its accumulation at division sites, where 68 it invaginates the OM through non-covalent interactions with newly-formed septal 69 peptidoglycan. 70 4 tol genes, which are found in most Gram-negative bacteria, were originally 71 identified by Luria and co-workers in the 1960s through mutations that engendered 72Escherichia coli tolerance towards colicins and filamentous bacteriophages 11 . 73Concomittant with this tolerance is a pleiotropic OM instability phenotype that is manifest 74 through cell filamentation and division defects, hypersensitivity towards detergents and 75 bile salts and leakage of periplasmic contents. The Tol assembly is essential in bacteria 76expressing O-antigens, is a virulence factor in host-pathogen interactions 12-14 and is 77 implicated in biofilm formation 15 . The core components of Tol are three IM proteins, 78TolQ, TolR and TolA, periplasmic TolB and peptidoglycan associated lipoprotein Pal in 79 the inner leaflet of the OM (Figure 1a) 3 . TolA in the inner membrane spans the 80 periplasm and undergoes PMF-driven conformational changes by virtue of its interaction 81 partners TolQ and TolR, which are homologues of the MotA and MotB stato...

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