Thematic review series: Glycerolipids. Acyltransferases in bacterial glycerophospholipid synthesis

Phospholipid biosynthesis is a vital facet of bacterial physiology that begins with the synthesis of the fatty acids by a soluble type II fatty acid synthase. The bacterial glycerol-phosphate acyltransferases utilize the completed fatty acid chains to form the first membrane phospholipid and thus play a critical role in the regulation of membrane biogenesis. The first bacterial acyltransferase described was PlsB, a glycerol-phosphate acyltransferase. PlsB is a key regulatory point that coordinates membrane phospholipid formation with cell growth and macromolecular synthesis. Phosphatidic acid is then produced by PlsC, a 1-acylglycerol-phosphate acyltransferase. These two acyltransferases use thioesters of either CoA or acyl carrier protein (ACP) as the acyl donors and have homologs that perform the same reactions in higher organisms. However, the most prevalent glycerol-phosphate acyltransferase in the bacterial world is PlsY, which uses a recently discovered acyl-phosphate fatty acid intermediate as an acyl donor. This unique activated fatty acid is formed from the acyl-ACP end products of the fatty acid biosynthetic pathway by PlsX, an acyl-ACP:phosphate transacylase.     

Bacterial fatty acid metabolism in modern antibiotic discovery

Bacterial fatty acid synthesis is essential for many pathogens and different from the mammalian counterpart. These features make bacterial fatty acid synthesis a desirable target for antibiotic discovery. The structural divergence of the conserved enzymes and the presence of different isozymes catalyzing the same reactions in the pathway make bacterial fatty acid synthesis a narrow spectrum target rather than the traditional broad spectrum target. Furthermore, bacterial fatty acid synthesis inhibitors are single-targeting, rather than multi-targeting like traditional monotherapeutic, broad-spectrum antibiotics. The single-targeting nature of bacterial fatty acid synthesis inhibitors makes overcoming fast-developing, target-based resistance a necessary consideration for antibiotic development. Target-based resistance can be overcome through multi-targeting inhibitors, a cocktail of single-targeting inhibitors, or by making the single targeting inhibitor sufficiently high affinity through a pathogen selective approach such that target-based mutants are still susceptible to therapeutic concentrations of drug. Many of the pathogens requiring new antibiotic treatment options encode for essential bacterial fatty acid synthesis enzymes. This review will evaluate the most promising targets in bacterial fatty acid metabolism for antibiotic therapeutics development and review the potential and challenges in advancing each of these targets to the clinic and circumventing target-based resistance. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.     

Improved method using muramic acid to estimate biomass of bacteria in sediments

Summary A method, which depends on the measurement of muramic acid content to estimate bacterial biomass, has been improved in sensitivity by two orders of magnitude. It is now applicable to any aquatic sediment, whereas previously it was mainly useful in the analysis of gut contents of deposit-feeding animals. Reduced NAD, a product of the oxidation of d-lactate derived from muramic acid, is assayed using bacterial luciferase. The amount of muramic acid in a number of terrestrial and marine bacteria was measured, and found to be lower than that obtained with the previous, less specific, assay procedure. The muramic acid content of a blue-green alga has been measured, thus allowing blue-green algae to be taken into account when estimating bacterial biomass. Experimental evidence is presented which shows that muramic acid in cell wall fragments of bacteria is rapidly degraded by microorganisms in a marine sediment.     

Endotoxic activity of complexes of myristic acid and proteins.

Complexes of myristic acid and bovine serum albumin, myristic acid and concanavalin A, beta-hydroxymyristic acid and concanavalin A, or dimethyl myristamide and concanavalin A are lethal for male BALB/c mice treated with mithramycin. Prior treatment of mice with myristic acid-protein complexes renders the animals resistant to a dose of bacterial endotoxin that is lethal for untreated animals. Prior treatment of mice with bacterial endotoxin renders them resistant to a combination of mithramycin and a complex of myristic acid and bovine serum albumin or dimethyl myristamide and concanvalin A that is lethal for untreated animals. These data indicate that a fatty acid is an important functional component of the endotoxin toxophore.     

Bacterial IMPDH gene used for the selection of mammalian cell transfectants

Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or require unique conditions. Here we describe a simple and versatile dominant selectable marker that involves bacterial IMP dehydrogenase (IMPDH), an enzyme essential for the replication of mammalian and bacterial cells. Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant to the toxic effect of the drug mycophenolic acid, which is an IMPDH inhibitor. We have demonstrated that transfection of human, monkey or Chinese hamster cell lines with an expression vector containing bacterial IMPDH and mycophenolic acid treatment resulted in the selection of colonies with a strikingly increased resistance to mycophenolic acid toxicity. Analysis of cells derived from these colonies indicated that the acquisition of this resistance was associated with bacterial IMPDH protein expression. As a proof of principle, we showed that mammalian cell transfection with a bicistronic IMPDH/GFP expression vector and mycophenolic acid treatment can be used to successfully select transfectants that express the fluorescent protein. These results indicate that bacterial IMPDH is a practical dominant selectable marker that can be used for the selection of transfectants that express exogenous genes or cDNAs in mammalian cells.     

Bacterial community associated with ensilage process of wilted guinea grass

Aims: To determine the effects of wilting, storage period and bacterial inoculant on the bacterial community and ensiling fermentation of guinea grass silage. Methods and Results: Fermentation products, colony counts and denaturing gradient gel electrophoresis (DGGE) profiles were determined. There was more lactic acid than acetic acid in all silages, but the lactic acid to acetic acid ratio decreased with storage time. This shift from lactic to acetic acid was not prevented even with a combination of wilting and bacterial inoculant. The DGGE analyses suggest that facultatively heterofermentative lactic acid bacteria (Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus pentosus) were involved in the shift to acetic acid fermentation. Conclusions: Lactic acid can dominate the fermentation in tropical grass silage with sufficient wilting prior to ensiling. Prolonged storage may lead to high levels of acetic acid without distinctive changes in the bacterial community. Significance and Impact of the Study: The bacterial community looks stable compared to fermentation products over the course of long storage periods in tropical grass silage. Acetic acid fermentation in tropical grass silage can be a result of the changes in bacterial metabolism rather than community structure.     

乙醇胁迫处理对酒酒球菌SD-2a生理特性的影响

为制备高效的葡萄酒乳酸菌发酵剂,以酒酒球菌SD-2a为试验材料,研究了5%、10%不同浓度的乙醇胁迫处理对菌体生长、细胞内MLE活性、冻干存活率及细胞膜脂肪酸组分的影响。试验结果表明乙醇胁迫处理明显降低了菌体的生长速率和生物产量。与对照相比,5%乙醇胁迫处理降低了菌体的冻干存活率,而10%乙醇处理却显著增加了菌体的冻干存活率。细胞膜脂肪酸组分的测定结果表明,前者细胞膜U/S比值为1.12,比对照降低了26.3%,而后者细胞膜U/S比值为2.29,比对照增加了50.6%。故认为酒酒球菌SD-2a为适应不同浓度的乙醇胁迫环境,在细胞膜脂肪酸水平上所采用的机制不同,且菌体的冻干存活率与其细胞膜脂肪酸组分密切相关。     

Role of Capsular Colanic Acid in Adhesion of Uropathogenic Escherichia coli

Urinary tract infections are the most common urologic disease in the United States and one of the most common bacterial infections of any organ system. Biofilms persist in the urinary tract and on catheter surfaces because biofilm microorganisms are resistant to host defense mechanisms and antibiotic therapy. The first step in the establishment of biofilm infections is bacterial adhesion; preventing bacterial adhesion represents a promising method of controlling biofilms. Evidence suggests that capsular polysaccharides play a role in adhesion and pathogenicity. This study focuses on the role of physiochemical and specific binding interactions during adhesion of colanic acid exopolysaccharide mutant strains. Bacterial adhesion was evaluated for isogenic uropathogenic Escherichia coli strains that differed in colanic acid expression. The atomic force microscope (AFM) was used to directly measure the reversible physiochemical and specific binding interactions between bacterial strains and various substrates as bacteria initially approach the interface. AFM results indicate that electrostatic interactions were not solely responsible for the repulsive forces between the colanic acid mutant strains and hydrophilic substrates. Moreover, hydrophobic interactions were not found to play a significant role in adhesion of the colanic acid mutant strains. Adhesion was also evaluated by parallel-plate flow cell studies in comparison to AFM force measurements to demonstrate that prolonged incubation times alter bacterial adhesion. Results from this study demonstrate that the capsular polysaccharide colanic acid does not enhance bacterial adhesion but rather blocks the establishment of specific binding as well as time-dependent interactions between uropathogenic E. coli and inert substrates.     

Role of capsular colanic acid in adhesion of uropathogenic Escherichia coli

Urinary tract infections are the most common urologic disease in the United States and one of the most common bacterial infections of any organ system. Biofilms persist in the urinary tract and on catheter surfaces because biofilm microorganisms are resistant to host defense mechanisms and antibiotic therapy. The first step in the establishment of biofilm infections is bacterial adhesion; preventing bacterial adhesion represents a promising method of controlling biofilms. Evidence suggests that capsular polysaccharides play a role in adhesion and pathogenicity. This study focuses on the role of physiochemical and specific binding interactions during adhesion of colanic acid exopolysaccharide mutant strains. Bacterial adhesion was evaluated for isogenic uropathogenic Escherichia coli strains that differed in colanic acid expression. The atomic force microscope (AFM) was used to directly measure the reversible physiochemical and specific binding interactions between bacterial strains and various substrates as bacteria initially approach the interface. AFM results indicate that electrostatic interactions were not solely responsible for the repulsive forces between the colanic acid mutant strains and hydrophilic substrates. Moreover, hydrophobic interactions were not found to play a significant role in adhesion of the colanic acid mutant strains. Adhesion was also evaluated by parallel-plate flow cell studies in comparison to AFM force measurements to demonstrate that prolonged incubation times alter bacterial adhesion. Results from this study demonstrate that the capsular polysaccharide colanic acid does not enhance bacterial adhesion but rather blocks the establishment of specific binding as well as time-dependent interactions between uropathogenic E. coli and inert substrates.     

Bacterial chemoattraction towards jasmonate plays a role in the entry of Dickeya dadantii through wounded tissues

Jasmonate is a key signalling compound in plant defence that is synthesized in wounded tissues. In this work, we have found that this molecule is also a strong chemoattractant for the phythopathogenic bacteria Dickeya dadantii (ex- Erwinia chysanthemi ). Jasmonic acid induced the expression of a subset of bacterial genes possibly involved in virulence/survival in the plant apoplast and bacterial cells pre-treated with jasmonate showed increased virulence in chicory and Saintpaulia leaves. We also showed that tissue wounding induced bacterial spread through the leaf surface. Moreover, the jasmonate-deficient aos1 Arabidopsis thaliana mutant was more resistant to bacterial invasion by D. dadantii than wild-type plants. These results are consistent with the hypothesis that sensing jasmonic acid by this bacterium helps the pathogen to ingress inside plant tissues.     

Tryptophan photoproduct(s): sensitized induction of strand breaks (or alkali-labile bonds) in bacterial deoxyribonucleic acid during near-ultraviolet irradiation.

Long-wavelength ultraviolet light (300 to 400 nm) converts L-tryptophan to a photoproduct that is toxic for bacterial cells in dark conditions. We now report that similar photoproducts of l-tryptophan sensitize bacterial deoxyribonucleic acid to 365-nm radiation, increasing the yield of deoxyribonucleic acid strand breaks (or alkali-labile bonds) by approximately 11.5-fold. Evidence is also presented which indicates that thse sensitized deoxyribonucleic acid lesions contribute to lethality for Escherichia coli irradiated with 365-nm ultraviolet light in suspensions of tryptophan photoproducts.     

A method for the estimation of bacterial biomass and community structure in mangrove-associated sediments

Many recent reports have proposed that certain monocarboxylic fatty acids found in sediments originate in the in situ bacterial population. In this study we have divided the acids derived from bacteria into nine subgroups, each characteristic of a distinct compositional group of bacteria. It is proposed that the abundance of selected marker acids from each bacterial subgroup (chemotype) can be used to estimate the biomass of that chemotype. Conversion factors from acid abundance to bacterial biomass have been estimated using literature data. Since this procedure results in nine biomass parameters, bacterial communities can be compared in terms of both total biomass and chemotype distribution, that is, biomass and community structure. The ability of this procedure to resolve community structure variations is illustrated with the interpretation of the fatty acid profiles of a spatially distributed set of mangrove-associated sediments.     

Indigenous butyric acid-degrading bacteria as surrogate pit latrine odour control: isolation,biodegradability performance and growth kinetics

Butyric acid is one of the volatile organic compounds that significantly contribute to malodour emission from pit latrines. The purpose of this work is to isolate and identify bacterial strains that have the capability to degrade butyric acid, determine their butyric acid degradation efficiencies and estimate their growth pattern parameters of microbiological relevance. Pure cultures of bacterial strains capable of degrading butyric acid were isolated from pit latrine faecal sludge using an enrichment technique and were identified based on 16S rRNA analysis. The bacterial strains were cultured in mineral salt medium (MSM) supplemented with 1000 mg L−1 butyric acid, as a sole carbon and energy source, at 30 ± 1 °C, pH 7 and 110 rpm under aerobic growth conditions. The modified Gompertz model was used to estimate growth pattern parameters of microbiological relevance. Bacterial strains were phylogenetically identified as Alcaligenes sp. strain SY1, Achromobacter animicus, Pseudomonas aeruginosa, Serratia marcescens, Achromobacter xylosoxidans, Bacillus cereus, Lysinibacillus fusiformis, Bacillus methylotrophicus and Bacillus subtilis. The bacterial strains in pure cultures degraded butyric acid of 1000 mg L−1 within 20–24 h. The growth kinetics of the bacterial strains in pure culture utilising butyric acid were well described by the modified Gompertz model. This work highlights the potential for use of these bacterial strains in microbial degradation of butyric acid for deodorisation of pit latrine faecal sludge. This work also contributes significantly to our understanding of bioremediation of faecal sludge odours and informs the development of appropriate odour control technologies that may improve odour emissions from pit latrines.     

Mutagenic effect of nalidixic acid on bacteriophage T4 (author's transl)

Nalidixic acid can efficiently induce the reversion of some T4 rII mutations. The great majority of the strains whose reversion can be induced by this antibiotic are also sensitive to the mutagenic action of proflavin, indicating that mutagenicity of nalidixic acid results in base pair addition or deletion. With bacterial host strains resistant to nalidixic acid, the mutagenic effect is greatly reduced but not the effects on phage multiplication. This fact shows that the mutagenic agent is not the nalidixic acid itself but a derivative synthesized in sensitive bacterial strains.     

Rhodococcus sp. strain CR-53 LipR, the first member of a new bacterial lipase family (family X) displaying an unusual Y-type oxyanion hole, similar to the Candida antarctica lipase clan

Bacterial lipases constitute the most important group of biocatalysts for synthetic organic chemistry. Accordingly, there is substantial interest in developing new valuable lipases. Considering the lack of information concerning the lipases of the genus Rhodococcus and taking into account the interest raised by the enzymes produced by actinomycetes, a search for putative lipase-encoding genes from Rhodococcus sp. strain CR-53 was performed. We isolated, cloned, purified, and characterized LipR, the first lipase described from the genus Rhodococcus. LipR is a mesophilic enzyme showing preference for medium-chain-length acyl groups without showing interfacial activation. It displays good long-term stability and high tolerance for the presence of ions and chemical agents in the reaction mixture. Amino acid sequence analysis of LipR revealed that it displays four unique amino acid sequence motifs that clearly separate it from any other previously described family of bacterial lipases. Using bioinformatics tools, LipR could be related only to several uncharacterized putative lipases from different bacterial origins, all of which display the four blocks of consensus amino acid sequence motifs that contribute to define a new family of bacterial lipases, namely, family X. Therefore, LipR is the first characterized member of the new bacterial lipase family X. Further confirmation of this new family of lipases was performed after cloning Burkholderia cenocepacia putative lipase, bearing the same conserved motifs and clustering in family X. Interestingly, all lipases grouping in the new bacterial lipase family X display a Y-type oxyanion hole, a motif conserved in the Candida antarctica lipase clan but never found among bacterial lipases. This observation contributes to confirm that LipR and its homologs belong to a new family of bacterial lipases.     

The role of indoleacetaldehyde in IAA production from tryptophan by plants and by their epiphytic bacteria

Tryptophan, tryptamine, or indolepyruvic acid were applied to 2 systems: a bacterial (pea stem sections containing the epiphytic bacteria) and a plant system (pea stem sections under sterile conditions). In the plant system, the production of indoleacetic acid and indoleethanol (tryptophol) from each applied indole derivative is clearly reduced by the aldehyde reagents bisulfite and dimedon, respectively. Indoleacetaldehyde is chromatographically detected after alkaline liberation from its bisulfite addition product. In the bacterial system, the production of indoleacetic acid and indoleethanol is likewise reduced by bisulfite and dimedon. However, after tryptophan or tryptamine application, we could not detect indoleacetaldehyde in the described way. In one case only, namely tryptamine application to the bacterial system, indoleethanol production (contrary to indoleacetic acid production) is scarcely reduced by the aldehyde reagents. This indicates a bacterial pathway tryptamine → indoleethanol which bypasses indoleacetaldehyde.     

“Just a spoonful of sugar...”: import of sialic acid across bacterial cell membranes

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.     

Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae

Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis.     

Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.