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Short-Duration Low-Direct-Current Electrical Field Treatment Is a Practical Tool for Considerably Reducing Counts of Gram-Negative Bacteria Entrapped in Gel Beads. R. Zvitov, 2004.Application of a direct-current electrical field for very short times can serve as a practical nonthermal procedure to reduce or modify the microbial distribution in gel beads . The viability of Escherichia coli and Serratia marcescens entrapped in alginate and agarose beads decreases as the field intensity and duration of electrical field increase . Residue R113 Is Essential for PhoP Dimerization and Function: a Residue Buried in the Asymmetric PhoP Dimer Interface Determined in the PhoPN Three-Dimensional Crystal Structure. Yinghua Chen, 2003.Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which Pi is growth limiting . Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes . DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3' of PhoP-binding consensus repeats in PhoP-repressed promoters . The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization . A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction . We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon . In vivo expression of either PhoPR113E, PhoPR113A, or PhoPD60K resulted in a Pho-negative phenotype . In vitro analysis showed that PhoPR113E was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize . Monomeric PhoPR113E  P was deficient in DNA binding, contributing to the PhoPR113E in vivo Pho-negative phenotype . While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential . Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function .
Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP-N-Acetylmuramate:Amino Acid Ligase Activity. Lars Hesse, 2003.Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans . Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity . The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis . However, chlamydiae do not synthesize detectable peptidoglycan . The paradox created by these observations is known as the chlamydial anomaly . The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity . In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E . coli mutant possessing a temperature-sensitive lesion in MurC . The recombinant MurC domain was overexpressed in and purified from E . coli . It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM) . Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable Vmax/Km values were obtained . Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule . However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide . Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of L-Valine. C. Lange, 2003.
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