Pathogenic leishmania secrete antigenically related chitinases which are encoded by a highly conserved gene locus

Recently, we identified and characterized a single-copy chitinase gene (LdCht1) from Leishmania donovani, a protozoan pathogen of humans. It has been hypothesized that this parasite enzyme plays a critical role in the survival of all Leishmania species within their sandfly vectors and for their transmission to humans. Thus, in the current study, pulse-field gel electrophoresis and Southern hybridization with the LdCht1 gene probe were used to demonstrate that this chitinase gene has been conserved across species lines of various pathogenic Leishmania. Further, immunoprecipitation and enzyme activity assays using an anti-LdCht1-peptide serum were used to show that the chitinases produced and released by this group of parasites possessed both highly conserved antigenic epitopes and enzyme activities. Results of these studies demonstrate that the chitinase gene locus and enzyme activity have been conserved across species lines among this group of human pathogens.     

A novel membrane-associated threonine permease encoded by the tdcC gene of Escherichia coli.

A novel L-threonine transport system is induced in Escherichia coli cells when incubated in amino acid-rich medium under anaerobic conditions. Genetic and biochemical analyses with plasmids harboring mutations in the anaerobically expressed tdcABC operon indicated that the tdcC gene product was responsible for L-threonine uptake. Competition experiments revealed that the L-threonine transport system is also involved in L-serine uptake and is partially shared for L-leucine transport; L-alanine, L-valine, and L-isoleucine did not affect L-threonine uptake. Transport of L-threonine was inhibited by the respiratory chain inhibitors KCN and carbonyl cyanide m-chlorophenylhydrazone and was Na+ independent. These results identify for the first time an E. coli gene encoding a permease specific for L-threonine-L-serine transport that is distinct from the previously described threonine-serine transport systems. A two-dimensional topological model predicted from the amino acid composition and hydropathy plot showed that the TdcC polypeptide appears to be an integral membrane protein with several membrane-spanning domains exhibiting a striking similarity with other bacterial permeases.     

Functions related to the receptor protein specified by the tsx gene of Escherichia coli

The receptor protein for the phage T6 and colicin K, coded by the tsx gene, facilitated the diffusion of all nucleosides and deoxynucleosides except cytidine and deoxcytidine through the outer membrane of Escherichia coli K-12 and Escherichia coli B. The tsx protein was coregulated with the nucleoside uptake system. Constitutive cytR and deoR mutants contained higher amounts of this protein than wild type strains. There was a good correlation between the initial rate of nucleoside uptake and the adsorption rate of phage T6. From the observation that nucleosides did not compete with each other in the translocation across the outer membrane and that they did not inhibit T6 adsorption it was concluded that the tsx protein forms a pore to which nucleosides have only little if any binding affinity.A major outer membrane protein specified by the ompA gene influenced the function of the tsx protein. Outer membranes of ompA mutants showed an enhanced binding of colicin K but the strains were colicin K insensitive (tolerant). The T6 phage adsorbed at the same rate and plated with the same efficiency as to ompA ⁺ strains. The uptake rate of thymidine and of adenosine was reduced by 16–33% in ompA mutants.The adsorption rate of phage T6 on mutants with altered lipopolysaccharide was the same or even higher than on wild type strains. However the plating efficiency was reduced ranging from 0–46%. Lipopolysaccharide plays no role in the primary adsorption of phage T6 but it is apparently required in a later step of the infection process.Non Standard Abbreviations LPS lipopolysaccharide - cAMP-CRP complex of cyclic adenosine 3,5-monophosphate (cAMP) and its receptor protein (CRP)     

Escherichia coli protein X is the recA gene product.

Escherichia coli protein X is known to be made in large amounts following DNA damage or inhibition of DNA replication. We have shown that it is identical to the recA gene product by partial proteolytic digestion of the radiochemically pure proteins and analysis by electrophoresis on polyacrylamide-sodium dodecyl sulfate gels.     

The Bacillus subtilis outB gene is highly homologous to an Escherichia coli ntr-like gene.

The Bacillus subtilis outB gene was found to have strong similarities to an Escherichia coli gene complementing ntr-like mutations in Rhodobacter capsulatus. The deduced gene products had 52% identical amino acids (65% similar residues). The phenotype of strains affected in the OutB function indicates that this B. subtilis gene may be involved in nitrogen utilization.     

The RNase Z homologue encoded by Escherichia coli elaC gene is RNase BN

In eukaryotes, archaea, and in some eubacteria, removal of 3' precursor sequences during maturation of tRNA is catalyzed by an endoribonuclease, termed RNase Z. In contrast, in Escherichia coli, a variety of exoribonucleases carry out final 3' maturation. Yet, E. coli retains an RNase Z homologue, ElaC, whose function is under active study. We have overexpressed and purified to homogeneity His-tagged ElaC and show here that it is, in fact, the previously described enzyme, RNase BN. Thus, purified ElaC displays structural and catalytic properties identical to those ascribed to RNase BN. In addition, an elaC mutant strain behaves identically to a known RNase BN- strain, CAN. Finally, we show that wild type elaC can complement the mutation in strain CAN and that the elaC gene in strain CAN carries a nonsense mutation that results in loss of RNase BN activity. These data correct a previous misassignment for the gene encoding RNase BN. Based on the fact that the original RNase BN mutation has now been identified, we propose that the elaC gene be renamed rbn.     

The distance between Escherichia coli genes is related to gene expression levels.

It is shown that the distance between Escherichia coli genes oriented in the same direction on the chromosome is positively correlated to the expression level of the downstream gene. It is suggested that this could be a strategy to avoid interference between ribosomes translating two different genes.     

The conserved active site motif A of Escherichia coli DNA polymerase I is highly mutable

Escherichia coli DNA polymerase I participates in DNA replication, DNA repair, and genetic recombination; it is the most extensively studied of all DNA polymerases. Motif A in the polymerase active site has a required role in catalysis and is highly conserved. To assess the tolerance of motif A for amino acid substitutions, we determined the mutability of the 13 constituent amino acids Val(700)-Arg(712) by using random mutagenesis and genetic selection. We observed that every residue except the catalytically essential Asp(705) can be mutated while allowing bacterial growth and preserving wild-type DNA polymerase activity. Hence, the primary structure of motif A is plastic. We present evidence that mutability of motif A has been conserved during evolution, supporting the premise that the tolerance for mutation is adaptive. In addition, our work allows identification of refinements in catalytic function that may contribute to preservation of the wild-type motif A sequence. As an example, we established that the naturally occurring Ile(709) has a previously undocumented role in supporting sugar discrimination.     

LETM1, a gene deleted in Wolf-Hirschhorn syndrome, encodes an evolutionarily conserved mitochondrial protein

The leucine zipper-, EF-hand-containing transmembrane protein 1 (LETM1) has recently been cloned in an attempt to identify genes deleted in Wolf-Hirschhorn syndrome (WHS), a microdeletion syndrome characterized by severe growth and mental retardation, hypotonia, seizures, and typical facial dysmorphic features. LETM1 is deleted in almost all patients with the full phenotype and has recently been suggested as an excellent candidate gene for the seizures in WHS patients. We have shown that LETM1 is evolutionarily conserved throughout the eukaryotic kingdom and exhibits homology to MDM38, a putative yeast protein involved in mitochondrial morphology. Using LETM1-EGFP fusion constructs and an anti-rat LetM1 polyclonal antibody we have demonstrated that LETM1 is located in the mitochondria. The present study presents information about a possible function for LETM1 and suggests that at least some (neuromuscular) features of WHS may be caused by mitochondrial dysfunction.     

Transformation of Rickettsia prowazekii to erythromycin resistance encoded by the Escherichia coli ereB gene

Rickettsia prowazekii, the etiologic agent of epidemic typhus, is an obligate, intracytoplasmic, parasitic bacterium. Recently, the transformation of this bacterium via electroporation has been reported. However, in these studies identification of transformants was dependent upon either selection of an R. prowazekii rpoB chromosomal mutation imparting rifampin resistance or expression of the green fluorescent protein and flow cytometric analysis. In this paper we describe the expression in R. prowazekii of the Escherichia coli ereB gene. This gene codes for an erythromycin esterase that cleaves erythromycin. To the best of our knowledge, this is the first report of the expression of a nonrickettsial, antibiotic-selectable gene in R. prowazekii. The availability of a positive selection for rickettsial transformants is an important step in the characterization of genetic analysis systems in the rickettsiae.