A cDNA previously shown to identify a salt-inducible root-specific transcript in Medicago sativa was used to screen an alfalfa library for the corresponding genomic sequence. One positive clone was recovered. The nucleotide sequence of a subclone contained a 329 bp 5' region upstream of the first ATG codon, a 1143 bp coding segment, and a 447 bp 3'-untranslated region interrupted by a single 475 bp intron. Translation of the coding segment, which was designated MsPRP2, suggested it encodes a chimeric 40,569 Da cell wall protein with an amino-terminal signal sequence, a repetitive proline-rich sequence, and a cysteine-rich carboxyl-terminal sequence homologous to nonspecific lipid transfer proteins. The 3'-untranslated region of MsPRP2 contained a sequence similar to one found to destabilize mRNAs transcribed from the elicitor-regulated proline-rich protein gene PvPRP1. Transcription run-on experiments using nuclei from salt-sensitive and salt-tolerant alfalfa callus suggested that the accumulation of MsPRP2 transcripts in salt-tolerant alfalfa cells grown in the presence of salt is due primarily to increased mRNA stability. The MsPRP2 gene thus may be a useful model for studying post-transcriptional salt-regulated expression of cell wall proteins.

Osmolyte accumulation and release can protect cells from abiotic stresses. In Escherichia coli, known mechanisms mediate osmotic stress-induced accumulation of K ؉ glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high-and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-N-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.

The taxonomic position of a novel halophilic endospore-forming bacterium previously isolated from a desert iguana was investigated by 16S rRNA gene sequencing. Comparative sequence analyses showed the unidentified bacterium to be phylogenetically loosely associated with some other spore-forming (Bacillus pantothenticus, Sporosarcina halophila) and non-spore-forming (Marinococcus albus) halotolerant bacteria. Based on the phenotypic and phylogenetic distinctiveness of the unidentified bacterium, it is proposed that it is classified in the genus Bacillus as a new species, Bacillus dipsosauri.

Significance and Impact of the Study: The enzyme urease is a virulence factor for the Gram-positive urinary tract pathogen Staphylococcus saprophyticus. We have shown that urease activity in cell-free extracts and whole bacterial cells is susceptible to inhibition by hydroxamates, phosphorodiamidates and flavonoids, but not by imidazoles. Acetohydroxamic acid and fluorofamide in particular can temporarily delay the increase in pH that occurs when Staph. saprophyticus is grown in an artificial urine medium. These results suggest that urease inhibitors may be useful as chemotherapeutic agents for the treatment of urinary tract infections caused by this micro-organism. AbstractUrease is a virulence factor for the Gram-positive urinary tract pathogen Staphylococcus saprophyticus. The susceptibility of this enzyme to chemical inhibition was determined using soluble extracts of Staph. saprophyticus strain ATCC 15305. Acetohydroxamic acid (K i = 8Á2 lg ml À1 = 0Á106 mmol l À1 ) and DL-phenylalanine hydroxamic acid (K i = 21 lg ml À1 = 0Á116 mmol l À1 ) inhibited urease activity competitively. The phosphorodiamidate fluorofamide also caused competitive inhibition (K i = 0Á12 lg ml À1 = 0Á553 lmol l À1 = 0Á000553 mmol l À1 ), but the imidazole omeprazole had no effect. Two flavonoids found in green tea extract [(+)-catechin hydrate (K i = 357 lg ml À1 = 1Á23 mmol l À1 ) and (À)-epigallocatechin gallate (K i = 210 lg ml À1 = 0Á460 mmol l À1 )] gave mixed inhibition. Acetohydroxamic acid, DL-phenylalanine hydroxamic acid, fluorofamide, (+)-catechin hydrate and (À)-epigallocatechin gallate also inhibited urease activity in whole cells of strains ATCC 15305, ATCC 35552 and ATCC 49907 grown in a rich medium or an artificial urine medium. Addition of acetohydroxamic acid or fluorofamide to cultures of Staph. saprophyticus in an artificial urine medium delayed the increase in pH that normally occurs during growth. These results suggest that urease inhibitors may be useful for treating urinary tract infections caused by Staph. saprophyticus.

~-Thiazolidine-4-carboxylate (T4C, y-thioproline) is a toxic analogue of L-proline. T4C can be oxidized by Escherichia coli to form N-formylcysteine, which is hydrolysed to yield formate and cysteine. To determine if L-proline dehydrogenase (EC 1.5.99.8) catalyses T4C degradation, membrane fractions from E. coli were tested for T4C and proline oxidation activity. The specific activity for T4C oxidation in membranes from bacteria grown with 10 mM-proline was similar to the specific activity for proline oxidation and about 100 times that in membranes from bacteria grown without proline. Both oxidation activities were inactivated at 45°C at the same rate. Membranes from a strain with a deletion of theputA gene encoding L-proline dehydrogenase or a strain with a putA::Tn5 insertion mutation had no detectable activity with either substrate. Although T4C was a simple competitive inhibitor of proline oxidation, proline inhibited T4C oxidation in a way that gave competitive but sigmoidal kinetics. At low concentrations, T4C induced proline dehydrogenase synthesis. Cysteine auxotrophs containing the putA::Tn5 mutation could still use T4C as a cysteine source, and bacteria with this mutation consumed oxygen in the presence of T4C at half the control rate. These results indicate that T4C is a substrate and an inducer of L-proline dehydrogenase but suggest that E. coli also contains a second enzyme catalysing T4C degradation.

Two independent mutants of Escherichia coli deficient in dipeptidyl carboxypeptidase activity (De)

L-Thiazolidine-4-carboxylate (T4C, thiaproline) is a sulfur-containing proline analog that stimulates the immune system in aging mice and inhibits urinary tract pathogens such as Escherichia coli. A constitutive NADP+-dependent T4C dehydrogenase activity was detected in the soluble fraction of a putA::Tn5 mutant of E. coli lacking l-proline dehydrogenase and partially purified by ammonium sulfate precipitation, dye-affinity chromatography on Cibacron Blue 3GA agarose, and ion-exchange chromatography on DEAE-cellulose. At each step in the purification, T4C dehydrogenase activity copurified with Delta1-pyrroline-5-carboxylate (P5C) reductase activity. E. coli strains with greatly reduced P5C reductase activity due to a proC mutation had no detectable T4C dehydrogenase activity. Although P5C reductase did not act on proline, it also catalyzed the oxidation of 3,4-dehydroproline. These results suggest that this biosynthetic enzyme may play a role in the degradation of proline analogs and limit the clinical efficacy of these compounds.

Aims: The aims of this study were to purify and characterize an extracellular a-amylase from the salt-tolerant bacterium Bacillus dipsosauri. Methods and Results: An extracellular a-amylase from B. dipsosauri strain DD1 was studied using the synthetic substrate 2-chloro-4-nitrophenyl-a-D-maltotrioside. Formation of the enzyme was induced by starch, repressed by D-glucose and highest after growth in medium containing 1AE0 mol l )1 KCl. The a-amylase activity increased with KCl concentration, showed a pH optimum of 6AE5, was stable up to 60°C and was stimulated by 1AE0 mol l )1 Na 2 SO 4 . The enzyme was purified from spent culture medium to apparent homogeneity by precipitation with ethanol, ion-exchange chromatography on DEAE-cellulose, centrifugal membrane filtration and gel-filtration chromatography on BioGel P-100. The purified enzyme had a denatured molecular mass of about 80 kDa but behaved on non-denaturing polyacrylamide gels as if it had a mass of about 30 kDa. The enzyme was partially inhibited by glucose-containing oligosaccharides of increasing length and strongly inhibited by the divalent cations Cd 2+ and Zn 2+ . Conclusions: The extracellular a-amylase from B. dipsosauri strain DD1 was purified to homogeneity and found to exhibit an unusually high degree of salt tolerance. Significance and Impact of the Study: The a-amylase from B. dipsosauri differs from previously described enzymes and may be useful for the processing of starches under high-salt conditions.