Comprehensive alanine-scanning mutagenesis of Escherichia coli CsrA defines two subdomains of critical functional importance

The RNA-binding protein CsrA (carbon storage regulator) of Escherichia coli is a global regulator of gene expression and is representative of the CsrA/RsmA family of bacterial proteins. These proteins act by regulating mRNA translation and stability and are antagonized by binding to small noncoding RNAs. Although the RNA target sequence and structure for CsrA binding have been well defined, little information exists concerning the protein requirements for RNA recognition. The three-dimensional structures of three CsrA/RsmA proteins were recently solved, revealing a novel protein fold consisting of two interdigitated monomers. Here, we performed comprehensive alanine-scanning mutagenesis on csrA of E. coli and tested the 58 resulting mutants for regulation of glycogen accumulation, motility, and biofilm formation. Quantitative effects of these mutations on expression of glgCA'-'lacZ, flhDC'-'lacZ, and pgaA'-'lacZ translational fusions were also examined, and eight of the mutant proteins were purified and tested for RNA binding. These studies identified two regions of the amino acid sequence that were critical for regulation and RNA binding, located within the first (beta1, residues 2-7) and containing the last (beta5, residues 40-47) beta-strands of CsrA. The beta1 and beta5 strands of opposite monomers lie adjacent and parallel to each other in the three-dimensional structure of this protein. Given the symmetry of the CsrA dimer, these findings imply that two distinct RNA binding surfaces or functional subdomains lie on opposite sides of the protein.     

Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication

Legionella pneumophila can replicate inside amoebae and also alveolar macrophages to cause Legionnaires' Disease in susceptible hosts. When nutrients become limiting, a stringent-like response coordinates the differentiation of L. pneumophila to a transmissive form, a process mediated by the two-component system LetA/S and the sigma factors RpoS and FliA. Here we demonstrate that the broadly conserved RNA binding protein CsrA is a global repressor of L. pneumophila transmission phenotypes and an essential activator of intracellular replication. By analysing csrA expression and the phenotypes of csrA single and double mutants and a strain that expresses csrA constitutively, we demonstrate that, during replication in broth, CsrA represses every post-exponential phase phenotype examined, including cell shape shortening, motility, pigmentation, stress resistance, sodium sensitivity, cytotoxicity and efficient macrophage infection. At the transition to the post-exponential phase, LetA/S relieves CsrA repression to induce transmission phenotypes by both FliA-dependent and -independent pathways. For L. pneumophila to avoid lysosomal degradation in macrophages, CsrA repression must be relieved by LetA/S before phagocytosis; conversely, before intracellular bacteria can replicate, CsrA repression must be restored. The reciprocal regulation of replication and transmission exemplified by CsrA likely enhances the fitness of microbes faced with fluctuating environments.     

Curli fibers are required for development of biofilm architecture in Escherichia coli K-12 and enhance bacterial adherence to human uroepithelial cells

Sessile bacteria show phenotypical, biochemical, and morphological differences from their planktonic counterparts. Curli, extracellular structures important for biofilm formation, are only produced at temperatures below 30 C in Escherichia coli K-12 strains. In this report, we show that E. coli K-12 can produce curli at 37 C when grown as a biofilm community. The curli-expressing strain formed more biofilms on polyurethane sheets than the curli-deficient strain under growth temperatures of both 25 C and 37 C. Curli are required for the formation of a three-dimensional mature biofilm, with characteristic water channels and pillars of bacteria. Observations by electron microscopy revealed the presence at the surfaces of the curli-deficient mutant in biofilm of flagella and type I pili. A wild-type curli-expressing E. coli strain significantly adhered to several lines of human uroepithelial cells, more so than an isogenic curlideficient strain. The finding that curli are expressed at 37 C in biofilm and enhance bacterial adherence to mammalian host cells suggests an important role for curli in pathogenesis.     

The pgaABCD locus of Escherichia coli promotes the synthesis of a polysaccharide adhesin required for biofilm formation

Production of a polysaccharide matrix is a hallmark of bacterial biofilms, but the composition of matrix polysaccharides and their functions are not widely understood. Previous studies of the regulation of Escherichia coli biofilm formation suggested the involvement of an unknown adhesin. We now establish that the pgaABCD (formerly ycdSRQP) locus affects biofilm development by promoting abiotic surface binding and intercellular adhesion. All of the pga genes are required for optimal biofilm formation under a variety of growth conditions. A pga-dependent cell-bound polysaccharide was isolated and determined by nuclear magnetic resonance analyses to consist of unbranched beta-1,6-N-acetyl-D-glucosamine, a polymer previously unknown from the gram-negative bacteria but involved in adhesion by staphylococci. The pga genes are predicted to encode envelope proteins involved in synthesis, translocation, and possibly surface docking of this polysaccharide. As predicted, if poly-beta-1,6-GlcNAc (PGA) mediates cohesion, metaperiodate caused biofilm dispersal and the release of intact cells, whereas treatment with protease or other lytic enzymes had no effect. The pgaABCD operon exhibits features of a horizontally transferred locus and is present in a variety of eubacteria. Therefore, we propose that PGA serves as an adhesin that stabilizes biofilms of E. coli and other bacteria.     

Global regulatory mutations in csrA and rpoS cause severe central carbon stress in Escherichia coli in the presence of acetate

The csrA gene encodes a small RNA-binding protein, which acts as a global regulator in Escherichia coli and other bacteria (T. Romeo, Mol. Microbiol. 29:1321-1330, 1998). Its key regulatory role in central carbon metabolism, both as an activator of glycolysis and as a potent repressor of glycogen biosynthesis and gluconeogenesis, prompted us to examine the involvement of csrA in acetate metabolism and the tricarboxylic acid (TCA) cycle. We found that growth of csrA rpoS mutant strains was very poor on acetate as a sole carbon source. Surprisingly, growth also was inhibited specifically by the addition of modest amounts of acetate to rich media (e.g., tryptone broth). Cultures grown in the presence of >/=25 mM acetate consisted substantially of glycogen biosynthesis (glg) mutants, which were no longer inhibited by acetate. Several classes of glg mutations were mapped to known and novel loci. Several hypotheses were examined to provide further insight into the effects of acetate on growth and metabolism in these strains. We determined that csrA positively regulates acs (acetyl-coenzyme A synthetase; Acs) expression and isocitrate lyase activity without affecting key TCA cycle enzymes or phosphotransacetylase. TCA cycle intermediates or pyruvate, but not glucose, galactose, or glycerol, restored growth and prevented the glg mutations in the presence of acetate. Furthermore, amino acid uptake was inhibited by acetate specifically in the csrA rpoS strain. We conclude that central carbon flux imbalance, inhibition of amino acid uptake, and a deficiency in acetate metabolism apparently are combined to cause metabolic stress by depleting the TCA cycle.     

大肠杆菌整体代谢网络调控基因(csrA)的敲除及其对苯丙氨酸生物合成的影响

csrA基因产物是大肠杆菌芳香族氨基酸生物合成途径中碳中心代谢有关的一种全局性调控蛋白质。采用Red敲除系统介导的同源重组的方法定位缺失大肠杆菌染色体csrA基因,经PCR、DNA测序等多种方法证实了基因重组缺失的可靠性。csrA基因缺失后,缺失菌株较对照菌株,糖酸转化率有所提高,发酵生产苯丙氨酸的能力也得到一定的提高,产酸提高约13%。