Distribution and purification of aspartate racemase in lactic acid bacteria

The distribution of aspartate racemase (EC 5.1.1.13) in various kinds of bacteria demonstrated that the enzyme occurs in lactic acid bacteria, such as Streptococcus species and Lactobacillus species. The enzyme from Streptococcus thermophilus IAM10064 was more thermostable than that from Streptococcus lactis IAM1198 which contained the enzyme most abundantly among the lactic acid bacteria we examined here. We purified the enzyme about 3400-fold to homogeneity from cell-free extract of S. thermophilus, which is composed of two identical subunits with a molecular weight of 28,000 as a homodimer. The enzyme utilizes specifically aspartate as a substrate, but not alanine and glutamate. Maximal reaction velocity was observed at 37 degrees C and around pH 8.0. The sequence of the NH2-terminal amino acids of the enzyme was determined to be Met-Glu-Asn-Phe-Phe-Ser-Ile-Leu-Gly-XXX-Met-Gly-Thr-Met-Ala-Thr-Glu-Ser- Phe-.     

Biochemical characterization of a novel lysine racemase from Proteus mirabilis BCRC10725

A lysine racemase gene (lyr) that consisted of an open reading frame of 1224-bp and encoded a protein with a calculated molecular mass of 45 kDa was cloned from the Proteus mirabilis BCRC10725 and expressed in Escherichia coli BL21(DE3). The purified His₆-tagged Lyr was most active towards lysine, exhibiting a specific activity of 2828 ± 97 U/mg. This enzyme also racemized arginine with a specific activity of 568 ± 28 U/mg but not other amino acids. The optimal conditions for Lyr activity to l-lysine were pH 8.0–9.0 and 50 °C. The racemization activity of Lyr was completely inhibited by 5 mM hydroxylamine and was partially restored by the addition of pyridoxal 5′-phosphate. The S394 residue of Lyr was subjected to site-directed mutagenesis. The arginine racemization activities of the S394Y, S394N, S394C and S394T variant proteins were increased by 1.5–1.8 fold compared to the wild-type Lyr, indicating that the S394 residue played a crucial role in determining the preference of Lyr to lysine and arginine.     

Activity of diaminopimelic acid decarboxylase in bacteria producing lysine

Activity of DAP-decarboxylase in lysine overproducing strains of Micrococcus luteus, M. varians and Arthrobacter globiformisLb reached the highest level during the end of the exponetial phase of growth and remained at the same high level during the stationary phase of growth when major bulk of lysine was accumulated. In comparison in a lysine non-producing strain of Arthrobacter globiformis I ₄ the activity of the same enzyme was low. DAP-decarboxylase of these three lysine overproducers has two specialities, persistence during the stationary phase and insensivity to repression by exogenous lysine.     

Exploring 9-benzyl purines as inhibitors of glutamate racemase (MurI) in Gram-positive bacteria

An early SAR study of a screening hit series has generated a series of 9-benzyl purines as inhibitors of bacterial glutamate racemase (MurI) with micromolar enzyme potency and improved physical properties. X-ray co-crystal EI structures were obtained.     

Distribution of polyamines in methanogenic bacteria

Members of all four families of methanogenic bacteria were analyzed for polyamine concentrations. High-performance liquid chromatography analysis of dansylated cell extracts revealed typical polyamine patterns for each family. Members of Methanobacteriaceae (family I) were characterized by very low polyamine concentrations; members of Methanococcaceae (family II) were characterized by putrescine and high spermidine concentrations; members of Methanomicrobiaceae (family III) were characterized by the presence of putrescine, spermidine, and sym-homospermidine; and members of Methanosarcinaceae (family IV) contained only high concentrations of sym-homospermidine in addition to putrescine. The highest polyamine concentration was found in Methanosarcina barkeri Jülich, with 0.35% putrescine in the dry cell material. The polyamine distribution found coincides with the dendrogram based on comparative cataloguing of 16S rRNA and offers a new, rapid chemotaxonomic method for characterizing methanogenic bacteria. Variation of the growth substrates (H2-CO2, methanol, acetate, and trimethylamine) for M. barkeri resulted in quantitative but not qualitative differences in polyamine composition.     

Distribution of plasmids in groundwater bacteria

Summary Bacteria isolated from groundwater aquifer core materials of pristine aquifers at Lula and Pickett, OK, and from a site with a history of aromatic hydrocarbon contamination and natural renovation located at Conroe, TX, were screened for the presence of plasmid DNA by alkaline or enzyme lysis and agarose gel techniques. Some of the isolates were also subjected to taxonomic tests in addition to screening for resistance to antibiotics, tolerance to heavy metal salts, and bacteriocin production. There was no significant difference in the distribution of the traits usually associated with plasmid occurrence in isolates from the three sites. These traits, which occurred at low frequencies, were not restricted to plasmid-bearing strains of the communities. Plasmids were found in isolates from all three sites, but on the average there was a significantly higher percentage of isolates containing plasmids in the samples from Conroe (19.4%) than from either Lula (1.8%) or Pickett (7.7%). The sizes of the plasmids found ranged between 3.5 and 202 kilobases but, for the Conroe samples, many more isolates (67%) contained smaller plasmids (<10 kb) rather than larger ones. No plasmids were found in bacteria recovered from naturally renovated aquifer material at the Conroe site.     

Distribution of IS5 in bacteria

Four different strains of Escherichia coli and several other bacteria were examined by Southern analysis for the presence of the insertion element IS5 and IS5-like sequences. Variations in the copy number, degree of homology and restriction pattern of the IS5-like sequence were found among the different organisms. The number and distribution of IS5 sequences do not appear to correlate with the evolutionary relationship of the bacteria in which they occur.     

Distribution of cytochromes in methanogenic bacteria

Abstract Various methanogenic bacteria belonging to the orders Methanobacteriales, Methanococcales , and Methanomicrobiales were examined for the presence of cytochromes. Those methanogens which are capable of growing only on H ₂+ CO ₂ or formate were found to lack cytochromes. However, membrane-bound cytochromes were detected in species able to utilize methanol, methylamines or acetate.     

Exploring 8-benzyl pteridine-6,7-diones as inhibitors of glutamate racemase (MurI) in gram-positive bacteria

A successful scaffold-hopping approach gave a novel series of inhibitors of bacterial glutamate racemase (MurI). Early SAR studies of the 8-benzyl pteridine-6,7-diones led to compounds with micromolar enzyme potency and antibacterial activity.     

Distribution of heterotrophic bacteria in Shira lake

The study of the horizontal and vertical distribution of heterotrophic bacteria in brackish Lake Shira in summer periods showed that mesophilic bacteria dominated in all areas of the lake, whereas psychrotolerant bacteria dominated in the metalimnion and hypolimnion of its central part. Nonhalophilic bacteria were mostly mesophilic and dominated in coastal waters. Most psychrotolerant bacteria were able to grow in the presence of 5-10% NaCl. Heterotrophic bacteria isolated in different regions of the lake were identified to a generic level. The isolates were classified into autochthonous and allochthonous microorganisms on the bases of their distribution pattern in the lake water, halotolerance, and ability to grow at low temperatures.     

Distribution of zinc-tolerant bacteria in stream sediments

The distribution of zinc-tolerant bacteria in sediments from three stream sites containing high (3125 g g^–1), medium (291 g g^–1), and low (109 g g^–1) concentrations of Zn was determined. Zinc tolerance was estimated by the ability of bacteria to grow on media amended with Zn concentrations ranging from 4 to 512 mg 1^–1. The presence of Zn-tolerant bacteria was correlated with the degree of heavy metal contamination; this correlation was more closely associated with readily extractable heavy metal concentrations than with the more rigorously extracted heavy metals. Low concentrations of Zn in media (4 to 16 mg 1^–1) were stimulatory to growth of bacteria from contaminated sites while concentrations as low as 4 mg 1^–1 were inhibitory to bacteria from the control site.     

Molecular cloning and nucleotide sequencing of the aspartate racemase gene from lactic acid bacteria Streptococcus thermophilus

The gene coding aspartate racemase (EC 5.1.1.13) was cloned from the lactic acid bacteria Streptococcus thermophilus IAM10064 and expressed efficiently in Escherichia coli. The 2.1 kilobase pairs long full length clone had an open reading frame of 729 nucleotides coding for 243 amino acids. The calculated molecular weight of 27,945 agreed well with the apparent molecular weight of 28,000 found in sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of the aspartate racemase purified from S. thermophilus. The N-terminal amino acid sequence from the purified protein exactly matches the derived sequence. In addition, the amino acid composition compiled from the derived sequence is very similar to that obtained from the purified recombinant protein. No significantly homologous proteins were found in a protein sequence data bank. Even the homology scores with alanine racemases of Salmonella typhimurium and Bacillus stearothermophilus were low. Aspartate racemase was overproduced in Escherichia coli NM522 with plasmid pAG6-2-7, which was constructed from two copies of the gene linked with a tac promoter and plasmid vector pUC18. The amount of aspartate racemase increases with the growth of E. coli and almost no degradation of the enzyme was observed. The maximum amount of the produced enzyme reached approx. 20% of the total protein of E. coli.     

Genome-wide analysis of lysine catabolism in bacteria reveals new connections with osmotic stress resistance

Lysine is catabolized via the saccharopine pathway in plants and mammals. In this pathway, lysine is converted to α-aminoadipic-δ-semialdehyde (AASA) by lysine-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH); thereafter, AASA is converted to aminoadipic acid (AAA) by α-aminoadipic-δ-semialdehyde dehydrogenase (AASADH). Here, we investigate the occurrence, genomic organization and functional role of lysine catabolic pathways among prokaryotes. Surprisingly, only 27 species of the 1478 analyzed contain the lkr and sdh genes, whereas 323 species contain aasadh orthologs. A sdh-related gene, identified in 159 organisms, was frequently found contiguously to an aasadh gene. This gene, annotated as lysine dehydrogenase (lysdh), encodes LYSDH an enzyme that directly converts lysine to AASA. Pipecolate oxidase (PIPOX) and lysine-6-aminotransferase (LAT), that converts lysine to AASA, were also found associated with aasadh. Interestingly, many lysdh–aasadh–containing organisms live under hyperosmotic stress. To test the role of the lysine-to-AASA pathways in the bacterial stress response, we subjected Silicibacter pomeroyi to salt stress. All but lkr, sdh, lysdh and aasadh were upregulated under salt stress conditions. In addition, lysine-supplemented culture medium increased the growth rate of S. pomeroyi under high-salt conditions and induced high-level expression of the lysdh–aasadh operon. Finally, transformation of Escherichia coli with the S. pomeroyi lysdh–aasadh operon resulted in increased salt tolerance. The transformed E. coli accumulated high levels of the compatible solute pipecolate, which may account for the salt resistance. These findings suggest that the lysine-to-AASA pathways identified in this work may have a broad evolutionary importance in osmotic stress resistance.