Production of L-Threonine by Mutants Resistant to Both α-Amino-β-hydroxyvaleric Acid and S-(2-Aminoethyl)-L-cysteine Derived from Brevibacterium flavum

Growth of Brevibacterium flavum FA-1-30 and FA-3-115, L-lysine producers derived from Br. flavum No. 2247 as S-(2-aminoethyl)-L-cysteine (AEC) resistant mutants, was inhibited by α-amino-β-hydroxyvaleric acid (AHV), and this inhibition was reversed by L-threonine. All the tested AHV resistant mutants derived from FA-1-30 accumulated more than 4 g/liter of L-threonine in media containing 10% glucose, and the best producer, FAB-44, selected on a medium containing 5 mg/ml of AHV produced about 15 g/liter of L-threonine. Many of AHV resistant mutants selected on a medium containing 2 mg/ml of AHV accumulated L-lysine as well as L-threonine, AHV resistant mutants derived from FA-3-115 produced 10.7 g/liter of L-threonine maximally. AEC resistant mutants derived from strains BB–82 and BB–69, which were L-threonine producers derived from Br. flavum No. 2247 as AHV resistant mutants, did not produce L-threonine more than the parental strains, and moreover, many of them did not accumulate L-threonine but L-lysine. Homoserine dehydrogenases of crude extracts from L-threonine producing AHV resistant mutants derived from FA–1–30 and FA–3–115 were insensitive to the inhibition by L-threonine, and those of L-threonine and L-lysine producing AHV resistant mutants from FA–1–30 were partially sensitive.

Correlation between L-threonine or L-lysine production and regulations of enzymatic activities of the mutants was discussed.     

Natural Phosphoryl and Acyl Variants of Lipid A from Neisseria meningitidis Strain 89I Differentially Induce Tumor Necrosis Factor-�� in Human Monocytes

The native lipooligosaccharide (LOS) from Neisseria meningitidis strain 89I was analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and the spectrum compared with that of the LOS after O-deacylation and hydrogen fluoride treatment. The data are consistent with the presence of natural variations in the LOS, which include a triphosphorylated lipid A (LA) with and without a phosphoethanolamine group, and both hexa- and pentaacylated LA molecules. Thin-layer chromatography was performed on 89I LA produced by hydrolysis of the LOS, and the purified LA molecules were analyzed by MALDI-TOF and tested for their relative ability to induce the secretion of tumor necrosis factor-α by human monocytic THP-1 cells and primary human monocytes. The potency of tumor necrosis factor-α induction varied by ∼2–10-fold, depending on the state of acylation and phosphorylation. The results highlight the significance of phosphorylation along with acylation of the LA component of LOS in stimulation of inflammatory signaling, and suggest that natural strain variation in these moieties may be a feature of meningococcal bacteria, which is of critical importance to the progression of the infection.The lipid A (LA)² portion of the lipopolysaccharide (LPS) or lipooligosaccharide (LOS) of Gram-negative bacteria is an inflammatory, pathogenic component of the bacterial outer membrane (1–3). Our interests lie in the Neisserial LA as it is implicated as a significant contributor to the pathogenesis of infections due to Neisseria meningitidis and Neisseria gonorrhoeae, which are of major public health concern around the world. In particular, N. meningitidis is the leading cause of epidemic meningitis and fatal sepsis in otherwise healthy individuals (4). On average more than 500,000 cases of meningococcal infection occur annually leading to ∼50,000 deaths, and large epidemic outbreaks can cause periodic spikes in occurrence. N. gonorrhoeae is a major cause of sexually transmitted infections, which can lead to pelvic inflammatory disease in 10–20% of infected women who can suffer from chronic pain, infertility, and ectopic pregnancy as a result (5). In addition, a growing number of studies have shown that gonococcal infection can facilitate the transmission of HIV (6).Numerous studies of LA signaling through the toll-like receptor 4 (TLR4) have increased our knowledge of the relationship between the LA structure and its inflammatory and immunogenic activity. The affinity of LA for monomeric binding to MD-2, which is a critical determinant in the agonist activity of LA for TLR4, has been found to be most potent in the hexaacylated compared with penta- or tetraacylated forms (7, 8). More recently, we and others have shown that triggering receptor expressed on myeloid cells-2 binds LOS and LPS in both myeloid and non-myeloid cells and initiates an inflammatory cytokine response (9, 10).Neisserial LOS lacks the repeating O-antigens of the LPS of the Gram-negative enteric bacteria, and differs also in the position, number, and chain length of the acyl groups on the LA. There can be differences in the acyl groups on the LA moieties within individual strains as well as between strains and species of Gram-negative bacteria (11, 12). The tetraacylated lipid IVa, which is a precursor of Escherichia coli LA has been found to be a TLR4 antagonist in human cells (13).In both LPS and LOS the number of phosphate (P) and phosphoethanolamine (PEA) groups on LA can vary (14), which impacts on the bioactivity of the molecule for innate immune responses. Recent work has shown that whereas hexaacyl monophosphoryl LA was restricted to the myeloid differentiation factor 88 (MyD88)-independent pathway, which resulted in T cell activation, the diphosphoryl LA also engaged the MyD88-dependent pathway, which activated NF-κB resulting in the production of TNF-α and other inflammatory cytokines (15). Because of its immunogenic properties, monophosphoryl LA has been approved for use as an adjuvant in a hepatitis B vaccine in Europe (16).We have observed major differences in the induction of proinflammatory cytokines through TLR4-mediated signaling elicited by various LOS purified from meningococcal and gonococcal strains (17). Little is known about the extent and biological significance of the natural structural variation in the LA of LOS occurring within a Neisserial strain. Recently, we reported that structural analyses of native LOS from different Neisserial strains indicated that differences in both acylation and phosphorylation of LA correlated significantly with the potency of LA to induce inflammatory cytokines (18). In this study, we used thin-layer chromatography (TLC) and matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) to investigate the heterogeneity and inflammatory activity of the structural variants of the LA from N. meningitidis strain 89I, the LOS of which we found previously to be the most potent inducer of TNF-α among a group of seven Neisserial strains studied (17).