Human milk oligosaccharides promote the growth of staphylococci

Human milk oligosaccharides (HMO), which constitute a major component of human milk, promote the growth of particular bacterial species in the infant's gastrointestinal tract. We hypothesized that HMO also interact with the bacterial communities present in human milk. To test this hypothesis, two experiments were conducted. First, milk samples were collected from healthy women (n = 16); culture-independent analysis of the bacterial communities was performed, HMO content was analyzed, and the relation between these factors was investigated. A positive correlation was observed between the relative abundance of Staphylococcus and total HMO content (r = 0.66). In a follow-up study, we conducted a series of in vitro growth curve experiments utilizing Staphylococcus aureus or Staphylococcus epidermidis and HMO isolated from human milk. HMO exhibited stimulatory effects on bacterial growth under various nutritional conditions. Analysis of culture supernatants from these experiments revealed that HMO did not measurably disappear from the culture medium, indicating that the growth-enhancing effects were not a result of bacterial metabolism of the HMO. Instead, stimulation of growth caused greater utilization of amino acids in minimal medium. Collectively, the data provide evidence that HMO may promote the growth of Staphylococcus species in the lactating mammary gland.     

Isolated cell behavior drives the evolution of antibiotic resistance

Bacterial antibiotic resistance is typically quantified by the minimum inhibitory concentration (MIC), which is defined as the minimal concentration of antibiotic that inhibits bacterial growth starting from a standard cell density. However, when antibiotic resistance is mediated by degradation, the collective inactivation of antibiotic by the bacterial population can cause the measured MIC to depend strongly on the initial cell density. In cases where this inoculum effect is strong, the relationship between MIC and bacterial fitness in the antibiotic is not well defined. Here, we demonstrate that the resistance of a single, isolated cell—which we call the single‐cell MIC (scMIC)—provides a superior metric for quantifying antibiotic resistance. Unlike the MIC, we find that the scMIC predicts the direction of selection and also specifies the antibiotic concentration at which selection begins to favor new mutants. Understanding the cooperative nature of bacterial growth in antibiotics is therefore essential in predicting the evolution of antibiotic resistance.     

Simulating Genetic Circuits in Bacterial Populations with Growth Heterogeneity

We computationally study genetic circuits in bacterial populations with heterogeneities in the growth rate. To that end, we present a stochastic simulation method for gene circuits in populations of cells and propose an efficient implementation that we call the “Next Family Method”. Within this approach, we implement different population setups, specifically Chemostat-type growth and growth in an ideal Mother Machine and show that the population structure and its statistics are different for the different setups whenever there is growth heterogeneity. Such dependence on the population setup is demonstrated, in the case of bistable systems with different growth rates in the stable states, to have distinctive signatures on quantities including the distributions of protein concentration and growth rates, and hysteresis curves. Applying this method to a bistable antibiotic resistance circuit, we find that as a result of the different statistics in different population setups, the estimated minimal inhibitory concentration of the antibiotic becomes dependent on the population setup in which it is measured.     

Isotopic labeling of c-type multiheme cytochromes overexpressed in E. coli

Progresses made in bacterial genome sequencing show a remarkable profusion of multiheme c-type cytochromes in many bacteria, highlighting the importance of these proteins in different cellular events. However, the characterization of multiheme cytochromes has been significantly retarded by the numerous experimental challenges encountered by researchers who attempt to overexpress these proteins, especially if isotopic labeling is required. Here we describe a methodology for isotopic labeling of multiheme cytochromes c overexpressed in Escherichia coli, using the triheme cytochrome PpcA from Geobacter sulfurreducens as a model protein. By combining different strategies previously described and using E. coli cells containing the gene coding for PpcA and the cytochrome c maturation gene cluster, an experimental labeling methodology was developed that is based on two major aspects: (i) use of a two-step culture growth procedure, where cell growth in rich media was followed by transfer to minimal media containing (15)N-labeled ammonium chloride, and (ii) incorporation of the heme precursor delta-aminolevulinic acid in minimal culture media. The yields of labeled protein obtained were comparable to those obtained for expression of PpcA in rich media. Proper protein folding and labeling were confirmed by UV-visible and NMR spectroscopy. To our knowledge, this is the first report of a recombinant multiheme cytochrome labeling and it represents a major breakthrough for functional and structural studies of multiheme cytochromes.     

Comparative analyses of transport proteins encoded within the genomes of Mycobacterium tuberculosis and Mycobacterium leprae

The co-emergence of multidrug resistant pathogenic bacterial strains and the Human Immunodeficiency Virus pandemic has made tuberculosis a leading public health threat. The causative agent is Mycobacterium tuberculosis (Mtu), a facultative intracellular parasite. Mycobacterium leprae (Mle), a related organism that causes leprosy, is an obligate intracellular parasite. Given that different transporters are required for bacterial growth and persistence under a variety of growth conditions, we conducted comparative analyses of transport proteins encoded within the genomes of these two organisms. A minimal set of genes required for intracellular and extracellular life was identified. Drug efflux systems utilizing primary active transport mechanisms have been preferentially retained in Mle and still others preferentially lost. Transporters associated with environmental adaptation found in Mtu were mostly lost in Mle. These findings provide starting points for experimental studies that may elucidate the dependencies of pathogenesis on transport for these two pathogenic mycobacteria. They also lead to suggestions regarding transporters that function in intra- versus extra-cellular growth.     

Rhamnolipid-dependent spreading growth of Pseudomonas aeruginosa on a high-agar medium: marked enhancement under CO2-rich anaerobic conditions

Anaerobiosis of Pseudomonas aeruginosa in infected organs is now gaining attention as a unique physiological feature. After anaerobic cultivation of P. aeruginosa wild type strain PAO1 T, we noticed an unexpectedly expanding colony on a 1.5% agar medium. The basic factors involved in this spreading growth were investigated by growing the PAO1 T strain and its isogenic mutants on a Davis high-agar minimal synthetic medium under various experimental conditions. The most promotive environment for this spreading growth was an O(2)-depleted 8% CO(2) condition. From mutational analysis of this spreading growth, flagella and type IV pili were shown to be ancillary factors for this bacterial activity. On the other hand, a rhamnolipid-deficient rhlA mutant TR failed to exhibit spreading growth on a high-agar medium. Complementation of the gene defect of the mutant TR with a plasmid carrying the rhlAB operon resulted in the restoration of the spreading growth. In addition, an external supply of rhamnolipid or other surfactants (surfactin from Bacillus subtilis or artificial product Tween 80) also restored the spreading growth of the mutant TR. Such activity of surfactants on bacterial spreading on a hard-agar medium was unique to P. aeruginosa under CO(2)-rich anaerobic conditions.     

Diversity and PAH growth abilities of bacterial strains isolated from a contaminated soil in Slovakia

Different abandoned industrial areas contaminated by polycyclic aromatic hydrocarbons (PAHs) are present in Slovakia. These environmental burdens are very dangerous to the health of human and environment. The bioremediation, based on the use of hydrocarbons degrading microorganisms, is a promising strategy to sanitize these polluted sites. The aim of this investigation was to assess the bacterial diversity of a PAHs-contaminated soil and to select the potential hydrocarbonoclastic bacteria which can be used for different bioremediation approaches. The bacterial strains were isolated on minimal medium agar supplemented with a mixture of PAHs. Seventy-three isolated strains were grouped by ribosomal interspacer analysis in 15 different clusters and representatives of each cluster were identified by 16S rRNA sequencing. The PAHs degradation abilities of all bacterial isolates were estimated by the 2,6-dichlorophenol indophenol assay and by their growth on minimal broth amended with a mixture of PAHs. Different kinds of strains, members of the genus Pseudomonas, Enterobacter, Bacillus, Arthrobacter, Acinetobacter and Sphingomonas, were isolated from the contaminated soil. Four isolates (Pseudomonas putida, Arthrobacter oxydans, Sphingomonas sp. and S. paucimobilis) showed promising PAHs-degrading abilities and therefore their possible employing in bioremediation strategies.     

Location, synthesis and function of glycolipids and polyglycerolphosphate lipoteichoic acid in Gram-positive bacteria of the phylum Firmicutes

Lipoteichoic acid (LTA) is a zwitterionic polymer found in the cell wall of many Gram-positive bacteria. A widespread and one of the best-studied forms of LTA consists of a polyglycerolphosphate (PGP) chain that is tethered to the membrane via a glycolipid anchor. In this review, we will summarize our current understanding of the enzymes involved in glycolipid and PGP backbone synthesis in a variety of different Gram-positive bacteria. The recent identification of key LTA synthesis proteins allowed the construction and analysis of mutant strains with defined defects in glycolipid or backbone synthesis. Using these strains, new information on the functions of LTA for bacterial growth, physiology and during developmental processes was gained and will be discussed. Furthermore, we will reintroduce the idea that LTA remains in close proximity to the bacterial membrane for its function during bacterial growth rather than as a surface-exposed structure.     

Growth of <Emphasis Type="Italic">E. coli</Emphasis> BL21 in minimal media with different gluconeogenic carbon sources and salt contents

Escherichia coli strain BL21 is commonly used as a host strain for protein expression and purification. For structural analysis, proteins are frequently isotopically labeled with deuterium (²H), ¹³C, or ¹⁵N by growing E. coli cultures in a medium containing the appropriate isotope. When large quantities of fully deuterated proteins are required, E. coli is often grown in minimal media with deuterated succinate or acetate as the carbon source because these are less expensive. Despite the widespread use of BL21, we found no data on the effect of different minimal media and carbon sources on BL21 growth. In this study, we assessed the growth behavior of E. coli BL21 in minimal media with different gluconeogenic carbon sources. Though BL21 grew reasonably well on glycerol and pyruvate, it had a prolonged lag-phase on succinate (20 h), acetate (10 h), and fumarate (20 h), attributed to the physiological adaptation of E. coli cells. Wild-type strain NCM3722 (K12) grew well on all the substrates. We also examined the growth of E. coli BL21 in minimal media that differed in their salt composition but not in their source of carbon. The commonly used M9 medium did not support the optimum growth of E. coli BL21 in minimal medium. The addition of ferrous sulphate to M9 medium (otherwise lacking it) increased the growth rate of E. coli cultures and significantly increased their cell density in the stationary phase. An erratum to this article can be found at     

Drying–Rewetting Cycles Affect Fungal and Bacterial Growth Differently in an Arable Soil

Drying and rewetting is a frequent physiological stress for soil microbial communities; a stress that is predicted to grow more influential with future climate change. We investigated the effect of repeated drying–rewetting cycles on bacterial (leucine incorporation) and fungal (acetate in ergosterol incorporation) growth, on the biomass concentration and composition (PLFA), and on the soil respiration. Using different plant material amendments, we generated soils with different initial fungal:bacterial compositions that we exposed to 6–10 repetitions of a drying–rewetting cycle. Drying–rewetting decreased bacterial growth while fungal growth remained unaffected, resulting in an elevated fungal:bacterial growth ratio. This effect was found irrespective of the initial fungal:bacterial biomass ratio. Many drying–rewetting cycles did not, however, affect the fungal:bacterial growth ratio compared to few cycles. The biomass response of the microbial community differed from the growth response, with fungal and total biomass only being slightly negatively affected by the repeated drying–rewetting. The discrepancy between growth- and biomass-based assessments underscores that microbial responses to perturbations might previously have been misrepresented with biomass-based assessments. In light of this, many aspects of environmental microbial ecology may need to be revisited with attention to what measure of the microbial community is relevant to study.     

Studies on the microflora associated with xenic cultures of Entamoeba gingivalis

The microflora associated with xenic stock cultures (ATCC 30927) of Entamoeba gingivalis, the major protozoan of the human oral cavity, were isolated and identified as Citrobacter diversus, Yersinia enterocolitica, Acinetobacter anitratus and Pseudomonas maltophilia. In studies to determine whether the bacterial isolates were able to utilize rice starch as a sole carbon source, Y. enterocolitica exhibited excellent growth in rice starch minimal medium and TYSGM-9 medium (with rice starch), but growth was weak in TYSGM-9 medium (without rice starch). C. diversus, A. anitratus and P. maltophilia exhibited poor growth in rice starch minimal medium, but they produced excellent growth in TYSGM-9 medium with or without rice starch. In order to determine the effect of the rice starch hydrolysis on Entamoeba growth, the filtrate from each isolate grown in rice starch minimal medium was added to an E. gingivalis culture grown in TYSGM-9 medium. The filtrate from a Y. enterocolitica culture grown in rice starch minimal medium enhanced E. gingivalis growth, but the filtrates from cultures of C. diversus, A. anitratus and P. maltophilia suppressed E. gingivalis growth. This supported the concept that Y. enterocolitica is capable of metabolizing rice starch into intermediate products, which in turn can be utilized by the amoeba.     

Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin

Growing cultures of an autolysis-defective pneumococcal mutant were exposed to [3H]benzylpenicillin at various multiples of the minimal inhibitory concentration and incubated until the growth of the cultures was halted. During the process of growth inhibition, we determined the rates and degree of acylation of the five penicillin-binding proteins (PBPs) and the rates of peptidoglycan incorporation, protein synthesis, and turbidity increase. The time required for the onset of the inhibitory effects of benzylpenicillin was inversely related to the concentration of the antibiotic, and inhibition of peptidoglycan incorporation always preceded inhibition of protein synthesis and growth. When cultures first started to show the onset of growth inhibition, the same characteristic fraction of each PBP was in the acylated form in all cases, irrespective of the antibiotic concentration. Apparently, saturation of one or more PBPs with the antibiotic beyond these threshold levels is needed to bring about interference with normal peptidoglycan production and cellular growth. Although it was not possible to correlate the inhibition of cell wall synthesis or cell growth with the degree of acylation (percentage saturation) of any single PBP, there was a correlation between the amount of peptidoglycan synthesized and the actual amount of PBP 2b that was not acylated. In cultures exposed to benzylpenicillin concentrations greater than eight times the minimal inhibitory concentration, the rates of peptidoglycan incorporation underwent a rapid decline when bacterial growth stopped. However, in cultures exposed to lower concentrations of benzylpenicillin (one to six times the minimal inhibitory concentration) peptidoglycan synthesis continued at constant rate for prolonged periods, after the turbidity had ceased to increase. We conclude that inhibition of bacterial growth does not require a complete inhibition or even a major decline in the rate of peptidoglycan incorporation. Rather, inhibition of growth must be caused by an as yet undefined process that stops cell division when the rate of incorporation of peptidoglycan (or synthesis of protein) falls below a critical value.     

Interactions of Photobleaching and Inorganic Nutrients in Determining Bacterial Growth on Colored Dissolved Organic Carbon

Abstract Bacteria are key organisms in the processing of dissolved organic carbon (DOC) in aquatic ecosystems. Their growth depends on both organic substrates and inorganic nutrients. The importance of allochthonous DOC, usually highly colored, as bacterial substrate can be modified by photobleaching. In this study, we examined how colored DOC (CDOC) photobleaching, and phosphorus (P) and nitrogen (N) availability, affect bacterial growth. Five experiments were conducted, manipulating nutrients (P and N) and sunlight exposure. In almost every case, nutrient additions had a significant, positive effect on bacterial abundance, production, and growth efficiency. Sunlight exposure (CDOC photobleaching) had a significant, positive effect on bacterial abundance and growth efficiency. We also found a significant, positive interaction between these two factors. Thus, bacterial use of CDOC was accelerated under sunlight exposure and enhanced P and N concentrations. In addition, the accumulation of cells in sunlight treatments was dependent on nutrient availability. More photobleached substrate was converted into bacterial cells in P- and N-enriched treatments. These results suggest nutrient availability may affect the biologically-mediated fate (new biomass vs respiration) of CDOC.     

Microalgal-facilitated bacterial oxidation of manganese

In the presence of unicellular microalgae, bacterial manganese oxidation was increased by up to ten times the rate produced by bacterial oxidation alone. Azide-poisoned controls demonstrated that the manganese-oxidizing bacteria were active in the algal-bacterial oxidation of manganese. Scanning electron microscopy showed that oxide formation occurred in a number of structurally different deposits on the surface of the alga. Studies involving algal cell fractionation showed that bacterial manganese oxidation was facilitated by the algal cell wall, possibly via Mn²⁺ adsorption. Variations in growth conditions had an effect on algal-bacterial oxide formation and composition. High nutrient (yeast extract, peptone and/or sucrose) levels favored microbial growth but lowered oxide formation, whereas optimal levels of manganese oxide formation required minimal media. High concentrations of either organic nutrients or mineral salts promoted manganese carbonate precipitation.     

A novel isotope labeling protocol for bacterially expressed proteins

Summary A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).     

Relationship between fitness and heterogeneity in exponentially growing microbial populations

Despite major environmental and genetic differences, microbial metabolic networks are known to generate consistent physiological outcomes across vastly different organisms. This remarkable robustness suggests that, at least in bacteria, metabolic activity may be guided by universal principles. The constrained optimization of evolutionarily motivated objective functions, such as the growth rate, has emerged as the key theoretical assumption for the study of bacterial metabolism. While conceptually and practically useful in many situations, the idea that certain functions are optimized is hard to validate in data. Moreover, it is not always clear how optimality can be reconciled with the high degree of single-cell variability observed in experiments within microbial populations. To shed light on these issues, we develop an inverse modeling framework that connects the fitness of a population of cells (represented by the mean single-cell growth rate) to the underlying metabolic variability through the maximum entropy inference of the distribution of metabolic phenotypes from data. While no clear objective function emerges, we find that, as the medium gets richer, the fitness and inferred variability for Escherichia coli populations follow and slowly approach the theoretically optimal bound defined by minimal reduction of variability at given fitness. These results suggest that bacterial metabolism may be crucially shaped by a population-level trade-off between growth and heterogeneity.     

Medium-dependent control of the bacterial growth rate

By combining results from previous studies of nutritional up-shifts we here re-investigate how bacteria adapt to different nutritional environments by adjusting their macromolecular composition for optimal growth. We demonstrate that, in contrast to a commonly held view the macromolecular composition of bacteria does not depend on the growth rate as an independent variable, but on three factors: (i) the genetic background (i.e. the strain used), (ii) the physiological history of the bacteria used for inoculation of a given growth medium, and (iii) the kind of nutrients in the growth medium. These factors determine the ribosome concentration and the average rate of protein synthesis per ribosome, and thus the growth rate. Immediately after a nutritional up-shift, the average number of ribosomes in the bacterial population increases exponentially with time at a rate which eventually is attained as the final post-shift growth rate of all cell components. After a nutritional up-shift from one minimal medium to another minimal medium of higher nutritional quality, ribosome and RNA polymerase syntheses are co-regulated and immediately increase by the same factor equal to the increase in the final growth rate. However, after an up-shift from a minimal medium to a medium containing all 20 amino acids, RNA polymerase and ribosome syntheses are no longer coregulated; a smaller rate of synthesis of RNA polymerase is compensated by a gradual increase in the fraction of free RNA polymerase, possibly due to a gradual saturation of mRNA promoters. We have also analyzed data from a recent publication, in which it was concluded that the macromolecular composition in terms of RNA/protein and RNA/DNA ratios is solely determined by the effector molecule ppGpp. Our analysis indicates that this is true only in special cases and that, in general, medium adaptation also depends on factors other than ppGpp.     

Pattern Formation by Competition: A Biological Example

We present a simple model based on a reaction-diffusion equation to explain pattern formation in a multicellular bacterium (Streptomyces). We assume competition for resources as the basic mechanism that leads to pattern formation; in particular we are able to reproduce the spatial pattern formed by bacterial aerial mycelium in the case of growth in minimal (low resources) and maximal (large resources) culture media.     

Competitive index in mixed infections: a sensitive and accurate assay for the genetic analysis of Pseudomonas syringae–plant interactions

Mixed infections have been broadly applied to the study of bacterial pathogens in animals. However, the application of mixed infection-based methods in plant pathogens has been very limited. An important factor for this limitation is the different dynamics that mixed infections have been reported to show in the different types of models. Reports in systemic animal infections have shown that any bacterium has the same probability of multiplying within a mixed infection than in a single infection. However, in plant pathogens, bacterial growth in a mixed infection does not seem to reflect growth in a single infection, as growth interference takes place between the co-inoculated strains. Here we show that growth interference in mixed infection between different Pseudomonas syringae strains is not intrinsic to growth within a plant host, but dependent on the dose of inoculation. We also show that the minimal inoculation dose required to avoid interference depends on the aggressiveness of the pathogen as well as the type of virulence factor that differentiates the co-inoculated strains. This study establishes the basis for the use of mixed infection-based applications to the study of phytopathogenic bacteria. Analysis of the virulence of a type III effector mutant and an hrp regulatory mutant illustrate the increased accuracy and sensitivity of competitive index assays vs. regular growth assays. Several applications of this assay are addressed, and potential implications for this and other mixed infection-based methods are discussed.     

Biochemical changes induced by salt stress in halotolerant bacterial isolates are media dependent as well as species specific

Halophilic bacteria respond to salt stress by regulating the cytosolic pools of organic solutes to achieve osmotic equilibrium. In order to understand the metabolic regulation of these organic solutes, for the first time, we have investigated the effect of salt on growth and biochemical changes in four major moderately halophilic bacterial strains isolated from a saltern region of the Kumta coast, India. The strains under study were Halomonas hydrothermalis VITP9, Bacillus aquimaris VITP4, Planococcus maritimus VITP21, and Virgibacillus dokdonensis VITP14, which exhibited similar salt tolerance (0% to 10% w/v NaCl) with optimal growth at 5% w/v NaCl. Biochemical analysis showed that the total intracellular organic solutes increased significantly with increasing NaCl concentration in the growth medium, and the compositions of the solutes were dependent on the type of strain and also on the nutrient richness of the growth medium. Glutamic acid levels increased in all the strains under salt stress, indicating the significance of glutamic acid as the anionic counterpart of K⁺/Na⁺ ions and precursor for other synthesized nitrogenous osmolytes. Though initial studies were performed with thin-layer chromatography, mass spectrometry was used to identify the major solutes accumulated by the strains under salt stress, such as proline (VITP4), ectoine (VITP14 and VITP9), and sugars (VITP21) under minimal medium and glycine betaine (by all the strains under study) under complex growth medium conditions. Such comparative study on the stress-dependent metabolic differences of different microbes, under identical experimental condition, helps to identify possible bacterial sources for the production of industrially important solutes.