Lactobacillus phospholipid molecular species were analyzed by mass spectrometry. Prominent anions were consistent with presence of the phosphatidylglycerols PG(37:2), PG(36:2), PG(35:1), PG(34:1), and PG(33:1). Diglycosyldiacylglycerol molecular species were also observed, although nitrogen-containing phospholipids were absent. An anion of m/z 759 was derived from an apparently novel type of lipid.Numerous studies have investigated the lipid composition of Lactobacillus spp. (8,19,21). Lactobacilli typically have n-C 16:0 , n-C 18:1 , and cyc-C 19 as the major carboxylate constituents, with lesser amounts of n-C 14:0 , n-C 16:1 , and n-C 18:0 and traces of odd-carbon-number acids in some species (6). Such fatty acid profiles are atypical of gram-positive groups but common in gram-positive bacteria (17). Other studies have analyzed polar lipids of lactobacilli and discovered that the vast majority of polar lipid is comprised of phosphatidylglycerol (PG) (8, 11), with smaller amounts of phosphatidic acid, diphosphatidylglycerol (cardiolipin), lysylphosphatidylglycerol (8, 10), phosphoglycolipids (10), and diglycosyldiacylglycerol (DGDG) (18).Virtually nothing is known of the distribution of individual molecular species within each type of phospholipid. In other groups of organisms, such an analysis has required expenditure of considerable time and effort to purify individual molecular species by chromatographic separation and then to analyze each molecular species in turn (2). An alternative approach, recently applied to the study of bacterial phospholipids (1, 3), is fast atom bombardment (FAB) mass spectrometry (MS) in which analysis of mixtures of molecular species is facilitated by the generation of ions retaining the intact molecular structure. FAB MS data for a single strain of Lactobacillus rhamnosus with respect to phospholipid molecular species have been published (7).The aim of this study was to determine the distribution of individual molecular species of phospholipids in a collection of Lactobacillus isolates.Fifteen Lactobacillus strains available from culture collections were analyzed (Table 1). All strains have been described previously (12, 13), and their identities were determined by using the API 50CHL test kit (API System, Vercieu, France) and the IDENTIFY computer program (5). Recent changes in the species assignments of some strains, not covered by the API 50CHL test kit for the identification of L. rhamnosus and L. paracasei subsp. paracasei (4), were incorporated into the computer program. For growth and harvesting, all strains were grown in 200-ml aliquots of MRS broth (Oxoid) for 18 to 24 h at 37ЊC as static batch cultures. Cultures were harvested by centrifugation and washed once in phosphate-buffered saline (pH 7.3). They were then washed in deionized water and freeze-dried. Phospholipid analysis was as follows. The methanol-chloroform extraction procedure, which used mechanical shaking at room temperature, was as described in a previous study (1). Phospholipid molecular species were sep...

The commercial antibiotic bicyclomycin (1) has been shown to target the essential transcriptional termination factor rho in Escherichia coli. Little is known, however, about the bicyclomycin binding site in rho. A recent structure−activity relationship study permitted us to design modified bicyclomycins that may irreversibly inactivate rho. The four compounds selected were C(5a)-(4-azidoanilino)dihydrobicyclomycin (3), C(5a)-(3-formylanilino)dihydrobicyclomycin (4), C(5)−norbicyclomycin C(5)-O-(4-azidobenzoate) (5), and C(5)-norbicyclomycin C(5)-O-(3-formylbenzoate) (6). In each of these compounds the inactivating unit was placed at the C(5)−C(5a) site in bicyclomycin. In compounds 3 and 5 an aryl azide moiety was used as a photoaffinity label whereas in 4 and 6 an aryl aldehyde group was employed as a reductive amination probe. The synthesis and spectral properties of 3−6 are described. Chemical studies demonstrated that 3 and 4 were stable in D2O and CD3OD (room temperature, 7 d), while 5 and 6 underwent significant change within 1 d. Biochemical investigations showed that 3 and 4 retained appreciable inhibitory activities in rho-dependent ATPase and transcription termination assays. In the ATPase assay, I 50 values for 1, 3, and 4 were 60, 135, and 70 μM, respectively. Correspondingly, the I 50 values for 5 and 6 were >400 and 225 μM, respectively. In the transcription termination assay, compounds 1, 3, and 4 all prevented (≥97%) the production of rho-dependent transcripts at 40 μM, whereas little (≤15%) inhibition of transcription termination was observed for 5 and 6 at this concentration. Antimicrobial evaluation of 3−6 showed that none of the four compounds exhibited antibiotic activity at 32 mg/mL or less against W3350 E. coli. The combined chemical and biochemical studies led to our further evaluation of 3 and 4. Photochemical irradiation (254 nm) of 3 in the presence of rho led to a 29−32% loss of rho ATPase activity. Attempts to confirm the irreversible adduction of 3 to rho by electrospray mass spectrometry were unsuccessful. No higher molecular weight adducts were detected. Incubation of rho with 4 at room temperature (4 h) followed by the addition of NaBH4 led to significant losses (>62%) of rho ATPase activity. Analyses of the 4−rho modified adduct showed appreciable levels of adduction (∼40%). Mass spectrometric analyses indicated a molecular weight for the adduct of approximately 47 410, consistent with a modification of a rho lysine residue by 4. Compound 4 was selected for additional studies.

The commercial antibiotic bicyclomycin (Bcm) has been shown to target the essential transcription termination factor Rho in Escherichia coli. Little is known about the Bcm binding domain in Rho. A recent structure-activity relationship study led us to evaluate the reductive amination probe, 5a-(3-formylanilino)dihydrobicyclomycin (FD-Bcm). Biochemical studies showed that FD-Bcm possessed inhibitory activities comparable to Bcm in Rho-dependent ATPase and transcription termination assays. Incubation of Rho with FD-Bcm, ATP, and poly(C) followed by NaBH 4 reduction and dialysis led to an appreciable loss of ATPase activity. Inclusion of Bcm with FD-Bcm in the reductive amination reaction protected Rho, indicating that Bcm and FD-Bcm competed for the same binding site in Rho. Incubation of Rho with FD-Bcm and poly(C) followed by NaBH 4 reduction provided a sample with residual ATPase activity (12%). Mass spectrometric analysis indicated the presence of two proteins in an approximate 1.2:1 ratio, whose masses corresponded to wild-type Rho (47,010 Da) and lysine-modified Rho (47,417 Da), respectively. Trypsin digestion of the Rho sample followed by high performance liquid chromatography separation and tandem mass spectrometry analysis identified the site of modification as Lys 181 within the combined tryptic fragment, Gly-Leu-Ile-Val-Ala-Pro-Pro-Lys-Ala-Gly-Lys (residues 174 -184). Similar analysis of a lesser modified sample (following incubation with inclusion of ATP) showed that addition had again occurred at Lys 181 . These findings provide the first structural information concerning the site of Bcm binding in Rho.Bicyclomycin (Bcm) 1 is a commercial antibiotic of novel structure (1-8). The primary site of action in Escherichia coli is unique and has been identified as the transcription termination factor, Rho (9). Rho is composed of six identical 47-kDa proteins of 419 amino acids (10) and exists in a planar, hexagonal (11-13) arrangement of proposed C 6 (14) or D 3 (15) geometry. Rho-dependent transcription termination sites occur at the ends of transcription units, at regulatory points before or between genes, and within genes (16). Transcription termination begins with the recognition and binding of Rho to discrete regions (rut sites) within the newly synthesized RNA chains (17). Binding is governed by a general requirement for a cytosine-rich sequence, and it extends across all six subunits of the functional protein, encompassing approximately 80 nucleotides (17)(18)(19)(20). Rho then extends its interaction toward the 3Ј end of the RNA polymerase elongation complex in a process coupled to ATP hydrolysis. The Rho-mediated release of RNA from the transcription bubble results from a destabilization of the complex, believed to be caused, in part, by the ATPase-dependent 5Ј 3 3Ј RNA-DNA helicase action of the protein (21).Details of the Bcm-Rho interaction have emerged in recent investigations (9,(22)(23)(24). Chemical studies have shown that nucleophilic amino acids readily react with Bcm, consistent with sever...

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