Calibrating the Bacterial Growth Rate Speedometer: A Re-evaluation of the Relationship Between Basal ppGpp, Growth, and RNA Synthesis in Escherichia coli.

The molecule guanosine tetraphophosphate (ppGpp) is most commonly considered an alarmone produced during acute stress. However, ppGpp is also present at low concentrations during steady-state growth. Whether ppGpp controls the same cellular targets at both low and high concentrations remains an open question and is vital for understanding growth rate regulation. It is widely assumed that basal ppGpp concentrations vary inversely with growth rate, and that the main function of basal ppGpp is to regulate transcription of ribosomal RNA in response to environmental conditions. Unfortunately, studies to confirm this relationship and to define regulatory targets of basal ppGpp are limited by difficulties in quantifying basal ppGpp. In this Perspective we compare reported concentrations of basal ppGpp in E. coli and quantify ppGpp within several strains using a recently developed analytical method. We find that although the inverse correlation between ppGpp and growth rate is robust across strains and analytical methods, absolute ppGpp concentrations do not absolutely determine RNA synthesis rates. In addition, we investigated the consequences of two separate RNA polymerase mutations that each individually reduce (but do not abolish) sensitivity to ppGpp and find that the relationship between ppGpp, growth rate, and RNA content of single-site mutants remains unaffected. Both literature and our new data suggest that environmental conditions may be communicated to RNA polymerase via an additional regulator. We conclude that basal ppGpp is one of potentially several agents controlling ribosome abundance and DNA replication initiation, but that evidence for additional roles in controlling macromolecular synthesis requires further study.

Posttranslational Control of PlsB Is Sufficient To Coordinate Membrane Synthesis with Growth in Escherichia coli.

Every cell must produce enough membrane to contain itself. However, the mechanisms by which the rate of membrane synthesis is coupled with the rate of cell growth remain unresolved. By comparing substrate and enzyme concentrations of the fatty acid and phospholipid synthesis pathways of Escherichia coli across a 3-fold range of carbon-limited growth rates, we show that the rate of membrane phospholipid synthesis during steady-state growth is determined principally through allosteric control of a single enzyme, PlsB. Due to feedback regulation of the fatty acid pathway, PlsB activity also indirectly controls synthesis of lipopolysaccharide, a major component of the outer membrane synthesized from a fatty acid synthesis intermediate. Surprisingly, concentrations of the enzyme that catalyzes the committed step of lipopolysaccharide synthesis (LpxC) do not differ across steady-state growth conditions, suggesting that steady-state lipopolysaccharide synthesis is modulated primarily via indirect control by PlsB. In contrast to steady-state regulation, we found that responses to environmental perturbations are triggered directly via changes in acetyl coenzyme A (acetyl-CoA) concentrations, which enable rapid adaptation. Adaptations are further modulated by ppGpp, which regulates PlsB activity during slow growth and growth arrest. The strong reliance of the membrane synthesis pathway upon posttranslational regulation ensures both the reliability and the responsiveness of membrane synthesis.IMPORTANCE How do bacterial cells grow without breaking their membranes? Although the biochemistry of fatty acid and membrane synthesis is well known, how membrane synthesis is balanced with growth and metabolism has remained unclear. This is partly due to the many control points that have been discovered within the membrane synthesis pathways. By precisely establishing the contributions of individual pathway enzymes, our results simplify the model of membrane biogenesis in the model bacterial species Escherichia coli Specifically, we found that allosteric control of a single enzyme, PlsB, is sufficient to balance growth with membrane synthesis and to ensure that growing E. coli cells produce sufficient membrane. Identifying the signals that activate and deactivate PlsB will resolve the issue of how membrane synthesis is synchronized with growth.

High mannose-specific lectin Msl mediates key interactions of the vaginal Lactobacillus plantarum isolate CMPG5300.

To characterize the interaction potential of the human vaginal isolate Lactobacillus plantarum CMPG5300, its genome was mined for genes encoding lectin-like proteins. cmpg5300.05_29 was identified as the gene encoding a putative mannose-binding lectin. Phenotypic analysis of a gene knock-out mutant of cmpg5300.05_29 showed that expression of this gene is important for auto-aggregation, adhesion to the vaginal epithelial cells, biofilm formation and binding to mannosylated glycans. Purification of the predicted lectin domain of Cmpg5300.05_29 and characterization of its sugar binding capacity confirmed the specificity of the lectin for high- mannose glycans. Therefore, we renamed Cmpg5300.05_29 as a mannose-specific lectin (Msl). The purified lectin domain of Msl could efficiently bind to HIV-1 glycoprotein gp120 and Candida albicans, and showed an inhibitory activity against biofilm formation of uropathogenic Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium. Thus, using a combination of molecular lectin characterization and functional assays, we could show that lectin-sugar interactions play a key role in host and pathogen interactions of a prototype isolate of the vaginal Lactobacillus microbiota.

Mass-Spectrometry-Based Quantification of Protein-Bound Fatty Acid Synthesis Intermediates from Escherichia coli.

The production of fatty acids from simple nutrients occurs via a complex biosynthetic pathway with dozens of intermediate compounds and multiple branch points. Despite its importance for microbial physiology and biotechnology, critical aspects of fatty acid biosynthesis, especially dynamics of in vivo regulation, remain poorly characterized. We have developed a liquid chromatography/mass spectroscopy (LC-MS) method for relative quantification of fatty acid synthesis intermediates in Escherichia coli, a model organism for studies of fatty acid metabolism. The acyl carrier protein, a vehicle for the substrates and intermediates of fatty acid synthesis, is extracted from E. coli, proteolytically digested, resolved using reverse-phase LC, and detected using electrospray ionization coupled with a tandem MS. Our method reliably resolves 21 intermediates of fatty acid synthesis, with an average relative standard deviation in ratios of individual acyl-ACP species to total ACP concentrations of 20%. We demonstrate that fast sampling and quenching of cells is essential to accurately characterize intracellular concentrations of ACP species. We apply our method to examine the rapid response of fatty acid metabolism to the antibiotic cerulenin. We anticipate that our method will enable the characterization of in vivo regulation and kinetics of microbial fatty acid synthesis at unprecedented detail and will improve integration of fatty acid synthesis into models of microbial metabolism.

Lectin-Like Molecules of Lactobacillus rhamnosus GG Inhibit Pathogenic Escherichia coli and Salmonella Biofilm Formation.

OBJECTIVES: Increased antibiotic resistance has catalyzed the research on new antibacterial molecules and alternative strategies, such as the application of beneficial bacteria. Since lectin molecules have unique sugar-recognizing capacities, and pathogens are often decorated with sugars that affect their survival and infectivity, we explored whether lectins from the probiotic strain Lactobacillus rhamnosus GG have antipathogenic properties. METHODS: The genome sequence of L. rhamnosus GG was screened for the presence of lectin-like proteins. Two genes, LGG_RS02780 and LGG_RS02750, encoding for polypeptides with an N-terminal conserved L-type lectin domain were detected and designated Llp1 (lectin-like protein 1) and Llp2. The capacity of Llp1 and Llp2 to inhibit biofilm formation of various pathogens was investigated. Sugar specificity was determined by Sepharose beads assays and glycan array screening. RESULTS: The isolated lectin domains of Llp1 and Llp2 possess pronounced inhibitory activity against biofilm formation by various pathogens, including clinical Salmonella species and uropathogenic E. coli, with Llp2 being more active than Llp1. In addition, sugar binding assays with Llp1 and Llp2 indicate specificity for complex glycans. Both proteins are also involved in the adhesion capacity of L. rhamnosus GG to gastrointestinal and vaginal epithelial cells. CONCLUSIONS: Lectins isolated from or expressed by beneficial lactobacilli could be considered promising bio-active ingredients for improved prophylaxis of urogenital and gastrointestinal infections.

Lactobacillus species as biomarkers and agents that can promote various aspects of vaginal health.

The human body is colonized by a vast number of microorganisms collectively referred to as the human microbiota. One of the main microbiota body sites is the female genital tract, commonly dominated by Lactobacillus spp., in approximately 70% of women. Each individual species can constitute approximately 99% of the ribotypes observed in any individual woman. The most frequently isolated species are Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii and Lactobacillus iners. Residing at the port of entry of bacterial and viral pathogens, the vaginal Lactobacillus species can create a barrier against pathogen invasion since mainly products of their metabolism secreted in the cervicovaginal fluid can play an important role in the inhibition of bacterial and viral infections. Therefore, a Lactobacillus-dominated microbiota appears to be a good biomarker for a healthy vaginal ecosystem. This balance can be rapidly altered during processes such as menstruation, sexual activity, pregnancy and various infections. An abnormal vaginal microbiota is characterized by an increased diversity of microbial species, leading to a condition known as bacterial vaginosis. Information on the vaginal microbiota can be gathered from the analysis of cervicovaginal fluid, by using the Nugent scoring or the Amsel's criteria, or at the molecular level by investigating the number and type of Lactobacillus species. However, when translating this to the clinical setting, it should be noted that the absence of a Lactobacillus-dominated microbiota does not appear to directly imply a diseased condition or dysbiosis. Nevertheless, the widely documented beneficial role of vaginal Lactobacillus species demonstrates the potential of data on the composition and activity of lactobacilli as biomarkers for vaginal health. The substantiation and further validation of such biomarkers will allow the design of better targeted probiotic strategies.