Partially Reciprocal Replacement of FlrA and FlrC in Regulation of Shewanella oneidensis Flagellar Biosynthesis

ABSTRACT In some bacteria with a polar flagellum, an established regulatory hierarchy controlling stepwise assembly of the organelle consists of four regulators: FlrA, σ 54 , FlrBC, and σ 28 . Because all of these regulators mediate the expression of multiple targets, they are essential to the assembly of a functional flagellum and therefore to motility. However, this is not the case for the gammaproteobacterium Shewanella oneidensis : cells lacking FlrB, FlrC, or both remain flagellated and motile. In this study, we unravel the underlying mechanism, showing that FlrA and FlrC are partially substitutable for each other in regulating flagellar assembly. While both regulators are bacterial enhancer binding proteins (bEBPs) for σ 54 , FlrA differs from FlrC in its independence of σ 54 for its own transcription and its inability to activate the flagellin gene flaA . These differences largely account for the distinct phenotypes resulting from the loss or overproduction of FlrA and FlrC. IMPORTANCE The assembly of a polar flagellum in bacteria has been characterized as relying on four regulators, FlrA, σ 54 , FlrBC, and σ 28 , in a hierarchical manner. They all are essential to the process and therefore to motility, except in S. oneidensis , in which FlrB, FlrC, or both together are not essential. Here we show that FlrA and FlrC, as bEBPs, are partially reciprocal in functionality in this species. As a consequence, the presence of one allows flagellar assembly and motility in the other's absence. Despite this, there are significant differences in the physiological roles played by these two regulators: FlrA is the master regulator of flagellar assembly, whereas FlrC fine-tunes motility. These intriguing observations open up a new avenue to further exploration of the regulation of flagellar assembly.

Molecular and Physiological Role of the Trehalose-Hydrolyzing α-Glucosidase from Thermus thermophilus HB27

ABSTRACT Trehalose supports the growth of Thermus thermophilus strain HB27, but the absence of obvious genes for the hydrolysis of this disaccharide in the genome led us to search for enzymes for such a purpose. We expressed a putative α-glucosidase gene (TTC0107), characterized the recombinant enzyme, and found that the preferred substrate was α,α-1,1-trehalose, a new feature among α-glucosidases. The enzyme could also hydrolyze the disaccharides kojibiose and sucrose (α-1,2 linkage), nigerose and turanose (α-1,3), leucrose (α-1,5), isomaltose and palatinose (α-1,6), and maltose (α-1,4) to a lesser extent. Trehalose was not, however, a substrate for the highly homologous α-glucosidase from T. thermophilus strain GK24. The reciprocal replacement of a peptide containing eight amino acids in the α-glucosidases from strains HB27 (LGEHNLPP) and GK24 (EPTAYHTL) reduced the ability of the former to hydrolyze trehalose and provided trehalose-hydrolytic activity to the latter, showing that LGEHNLPP is necessary for trehalose recognition. Furthermore, disruption of the α-glucosidase gene significantly affected the growth of T. thermophilus HB27 in minimal medium supplemented with trehalose, isomaltose, sucrose, or palatinose, to a lesser extent with maltose, but not with cellobiose (not a substrate for the α-glucosidase), indicating that the α-glucosidase is important for the assimilation of those four disaccharides but that it is also implicated in maltose catabolism.

Differences in the damage caused by glaze ice on codominant Acer saccharum and Fagus grandifolia

A severe glaze ice storm had greater destructive impact on Fagus grandifolia than on codominant Acer saccharum trees in a mature southern Quebec forest. Both the numbers and total biomass of major branches lost by beech were significantly greater than by sugar maple compared with the contribution of each species to the canopy composition. This greater ice damage to beech suggests that reciprocal replacement processes involving beech and maple seedlings cannot completely account for the maintenance of beech–maple codominance in northern forests subject to relatively frequent ice storms. We hypothesize that the ability of beech to root sprout is important in compensating for its greater susceptibility to ice damage and contributes to the maintenance of beech–maple codominance in northern forests.

Effects of Composition of Diluent, Method of Addition of Glycerol, Freezing Rate, and Storage Temperature on the Revival of Ram Spermatozoa After Deep-Freezing

The addition of 6% (vJv) egg yolk to a synthetic diluent [247 mM glucose, 49 mM NaCI, 5 mM KCI, 5 mM phosphate buffer, and 7�5% (vJv) glycerol] improved the survival of ram spermatozoa after deep�freezing. Judging by the activity of spermatozoa after thawing, a single addition of glycerol to the diluted semen at 5�C was as effective as multiple additions giving a graded increase of glycerol concentra-tion over a period of 20 min. Reciprocal replacement of the sodium chloride of the diluent by an increase in the concentration of the phosphate buffer showed that motility of the spermatozoa after thawing was depressed when the level of phosphate exceeded 10 mM.