Identification and molecular analysis of PcsB, a protein required for cell wall separation of group B streptococcus.

Group B streptococcus (GBS) is the leading cause of bacterial sepsis and meningitis in neonates. N-terminal sequencing of major proteins in the culture supernatant of a clinical isolate of GBS identified a protein of about 50 kDa which could be detected in all of 27 clinical isolates tested. The corresponding gene, designated pcsB, was isolated from a GBS cosmid library and subsequently sequenced. The deduced PcsB polypeptide consists of 447 amino acid residues (M(r), 46,754), carries a potential N-terminal signal peptide sequence of 25 amino acids, and shows significant similarity to open reading frames of unknown function from different organisms and to the murein hydrolase P45 from Listeria monocytogenes. Northern blot analysis revealed a monocistronic transcriptional organization for pcsB in GBS. Insertional inactivation of pcsB in the genome of GBS resulted in mutant strain Sep1 exhibiting a drastically reduced growth rate compared to the parental GBS strain and showing an increased susceptibility to osmotic pressure and to various antibiotics. Electron microscopic analysis of GBS mutant Sep1 revealed growth in clumps, cell separation in several planes, and multiple division septa within single cells. These data suggest a pivotal role of PcsB for cell division and antibiotic tolerance of GBS.

Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells.

Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce epithelial cell actin rearrangements resulting in pedestal formation beneath adherent bacteria. This requires the secretion of specific virulence proteins needed for signal transduction and intimate adherence. EPEC interaction induces tyrosine phosphorylation of a protein in the host membrane, Hp90, which is the receptor for the EPEC outer membrane protein, intimin. Hp90-intimin interaction is essential for intimate attachment and pedestal formation. Here, we demonstrate that Hp90 is actually a bacterial protein (Tir). Thus, this bacterial pathogen inserts its own receptor into mammalian cell surfaces, to which it then adheres to trigger additional host signaling events and actin nucleation. It is also tyrosine-phosphorylated upon transfer into the host cell.

Regulation of acetate metabolism in Corynebacterium glutamicum: transcriptional control of the isocitrate lyase and malate synthase genes.

In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes (aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum.

Enteropathogenic E. coli exploitation of host epithelial cells.

Enteropathogenic E. coli (EPEC) is a leading cause of neonatal diarrhea worldwide. These organisms adhere to the intestinal cell surface, causing rearrangement in the epithelial cell surface and underlying cytoskeleton, resulting in a structure termed an attaching/effacing (A/E) lesion. A/E lesion formation is thought necessary for EPEC-mediated disease. EPEC secretes several proteins that trigger signal transduction, intimate adherence, and cytoskeletal rearrangements in epithelial cells. Additionally, it produces intimin, an outer membrane product that mediates intimate adherence. Together these various bacterial molecules contribute to the intimate relationship that is formed by EPEC with host epithelial cells which results in A/E lesion formation and diarrhea.

A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation.

Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce actin accumulation beneath adherent bacteria. We found that EPEC adherence to epithelial cells mediates the formation of fingerlike pseudopods (up to 10 microm) beneath bacteria. These actin-rich structures also contain tyrosine phosphorylated host proteins concentrated at the pseudopod tip beneath adherent EPEC. Intimate bacterial adherence (and pseudopod formation) occurred only after prior bacterial induction of tyrosine phosphorylation of an epithelial membrane protein, Hp90, which then associates directly with an EPEC adhesin, intimin. These interactions lead to cytoskeletal nucleation and pseudopod formation. This is the first example of a bacterial pathogen that triggers signals in epithelial cells which activates receptor binding activity to a specific bacterial ligand and subsequent cytoskeletal rearrangement.

Malate synthase from Corynebacterium glutamicum: sequence analysis of the gene and biochemical characterization of the enzyme.

Malate synthase is one of the key enzymes of the glyoxylate cycle and is essential for growth on acetate as sole carbon source. The aceB gene from Corynebacterium glutamicum, encoding malate synthase, was isolated, subcloned and expressed in Escherichia coli and C. glutamicum. Sequencing of a 3024 bp DNA fragment containing the aceB gene revealed that it is located close to the isocitrate lyase gene aceA. The two genes are separated by 597 bp and are transcribed in divergent directions. The predicted aceB gene product consists of 739 amino acids with an M(r) of 82,362. Interestingly, this polypeptide shows only weak identity with malate synthase polypeptides from other organisms and possesses an extra N-terminal sequence of about 170 amino acid residues. Inactivation of the chromosomal aceB gene led to the absence of malate synthase activity and to the inability to grow on acetate, suggesting that only one malate synthase is present in C. glutamicum. The malate synthase was purified from an aceB-overexpressing C. glutamicum strain and biochemically characterized. The native enzyme was shown to be a monomer migrating at an M(r) of about 80,000. By sequencing the N-terminus of malate synthase the predicted translational start site of the enzyme was confirmed. The enzyme displayed Km values of 30 microM and 12 microM for the substrates glyoxylate and acetyl CoA, respectively. Oxalate, glycolate and ATP were found to be inhibitors of malate synthase activity. The present study provides evidence that the malate synthase from C. glutamicum is functionally similar to other malate synthase enzymes but is different both in size and primary structure.

Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme.

Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anaplerotic enzyme for growth on acetate as a carbon source. It is assumed to be of major importance in carbon flux control in the amino acid-producing organism Corynebacterium glutamicum. In crude extracts of C. glutamicum, the specific activities of isocitrate lyase were found to be 0.01 U/mg of protein after growth on glucose and 2.8 U/mg of protein after growth on acetate, indicating tight regulation. The isocitrate lyase gene, aceA, was isolated, subcloned, and characterized. The predicted gene product of aceA consists of 432 amino acids (M(r), 47,228) and shows up to 57% identity to the respective enzymes from other organisms. Downstream of aceA, a gene essential for thiamine biosynthesis was identified. Overexpression of aceA in C. glutamicum resulted in specific activities of 0.1 and 7.4 U/mg of protein in minimal medium containing glucose and acetate, respectively. Inactivation of the chromosomal aceA gene led to an inability to grow on acetate and to the absence of any detectable isocitrate lyase activity. Isocitrate lyase was purified to apparent homogeneity and subjected to biochemical analysis. The native enzyme was shown to be a tetramer of identical subunits, to exhibit an ordered Uni-Bi mechanism of catalysis, and to be effectively inhibited by 3-phosphoglycerate, 6-phosphogluconate, phosphoenolpyruvate, fructose-1,6-bisphosphate, and succinate.

Stable Expression of hom-1-thrB in Corynebacterium glutamicum and Its Effect on the Carbon Flux to Threonine and Related Amino Acids.

The hom-1-thrB operon encodes homoserine dehydrogenase resistant to feedback inhibition by L-threonine and homoserine kinase. Stable expression of this operon has not yet been attained in different Corynebacterium glutamicum strains. We studied the use of chromosomal integration and of a low-copy-number vector for moderate expression of the hom-1-thrB operon to enable an analysis of the physiological consequences of its expression in C. glutamicum. Strains carrying one, two, or three copies of hom-1-thrB were obtained. They showed proportionally increased enzyme activity of feedback-resistant homoserine dehydrogenase and of homoserine kinase. This phenotype was stably maintained in all recombinants for more than 70 generations. In a lysine-producing C. glutamicum strain which does not produce any threonine, expression of one copy of hom-1-thrB resulted in the secretion of 39 mM threonine. Additional copies resulted in a higher, although not proportional, accumulation of threonine (up to 69 mM). This indicates further limitations of threonine production. As the copy number of hom-1-thrB increased, increasing amounts of homoserine (up to 23 mM) and isoleucine (up to 34 mM) were secreted. Determination of the cytosolic concentration of the respective amino acids revealed an increase of intracellular threonine from 9 to 100 mM and of intracellular homoserine from 4 to 74 mM as the copy number of hom-1-thrB increased. These results suggest that threonine production with C. glutamicum is limited by the efflux system for this amino acid. Furthermore, the results show the successful use of moderate and stable hom-1-thrB expression for directing the carbon flux from aspartate to threonine.

Discrimination of Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactofermentum by restriction pattern analysis of DNA adjacent to the hom gene.

Different strains of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum were analysed for restriction fragment length polymorphism using the homoserine dehydrogenase gene (hom) as a probe. The hybridization patterns obtained PvuII- or Asp700-restriction of chromosomal DNA were specific and distinguishable for each of the three species and identical for the different strains of each species. Thus, the method employed allows rapid distinction of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum. The former species could also be discriminated from the latter two by its resistance to 0.5 g/l of the methionine analog ethionine.

Analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase.

From a Corynebacterium glutamicum mutant possessing a homoserine dehydrogenase resistant to feedback inhibition by L-threonine, the corresponding gene (homFBR) was analyzed and compared with the wild-type hom gene. DNA fragment exchange experiments between both genes showed that a 0.23-kb region close to the 3' terminus of homFBR was responsible for deregulation. Nucleotide sequence analysis revealed a single transition from G to A in homFBR leading to replacement of glycine-378 by glutamate in the mutant homoserine dehydrogenase.