Glucosyltransferase phase variation in Streptococcus gordonii modifies adhesion to saliva-coated hydroxyapatite surfaces in a sucrose-independent manner

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72317/1/j.1399-302X.1992.tb00521.x.pd

Molecular analysis of representative Streptococcus gordonii Spp phase variants reveals no differences in the glucosyltransferase structural gene, gtfG

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73398/1/j.1399-302X.1997.tb00622.x.pd

Sortase A Substrate Specificity in GBS Pilus 2a Cell Wall Anchoring

Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtAΔN40) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtAΔN40 does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus

Identifying Low pH Active and Lactate-Utilizing Taxa within Oral Microbiome Communities from Healthy Children Using Stable Isotope Probing Techniques

<div><h3>Background</h3><p>Many human microbial infectious diseases including dental caries are polymicrobial in nature. How these complex multi-species communities evolve from a healthy to a diseased state is not well understood. Although many health- or disease-associated oral bacteria have been characterized <em>in vitro</em>, their physiology within the complex oral microbiome is difficult to determine with current approaches. In addition, about half of these species remain uncultivated to date with little known besides their 16S rRNA sequence. Lacking culture-based physiological analyses, the functional roles of uncultivated species will remain enigmatic despite their apparent disease correlation. To start addressing these knowledge gaps, we applied a combination of Magnetic Resonance Spectroscopy (MRS) with RNA and DNA based Stable Isotope Probing (SIP) to oral plaque communities from healthy children for <em>in vitro</em> temporal monitoring of metabolites and identification of metabolically active and inactive bacterial species.</p> <h3>Methodology/Principal Findings</h3><p>Supragingival plaque samples from caries-free children incubated with ¹³C-substrates under imposed healthy (buffered, pH 7) and diseased states (pH 5.5 and pH 4.5) produced lactate as the dominant organic acid from glucose metabolism. Rapid lactate utilization upon glucose depletion was observed under pH 7 conditions. SIP analyses revealed a number of genera containing cultured and uncultivated taxa with metabolic capabilities at pH 5.5. The diversity of active species decreased significantly at pH 4.5 and was dominated by <em>Lactobacillus</em> and <em>Propionibacterium</em> species, both of which have been previously found within carious lesions from children.</p> <h3>Conclusions/Significance</h3><p>Our approach allowed for identification of species that metabolize carbohydrates under different pH conditions and supports the importance of Lactobacilli and Propionibacterium in the development of childhood caries. Identification of species within healthy subjects that are active at low pH can lead to a better understanding of oral caries onset and generate appropriate targets for preventative measures in the early stages.</p> </div

Amino acid requirements of Streptococcus mutans and other oral streptococci

The amino acid requirements of Streptococcus mutans strains AHT, OMZ-61, FA-1, BHT, GS-5, JC-2, Ingbritt, At6T, OMZ-176, 6715, Streptococcus salivarius HHT, Streptococcus sanguis OMZ-9, and strain 72x46 were determined in a chemically defined medium. When grown anaerobically in the presence of sodium carbonate (or bicarbonate for a few strains), few amino acids were required. All strains tested required cystine (or cystine) as a nutrient. Three strains (S. mutans OMZ-176, FA-1, and BHT) required glutamate (and/or glutamine). A third amino acid (lysine for S. mutans FA-1 and histidine for S. mutans OMZ-176) was required by two of the three strains which required glutamate. The amino acids mentioned above were required for all conditions of incubation (and inoculum) tested. The requirements for several other amino acids were conditional, that is, dependent on the incubation conditions and inoculum used. For example, when carbonate was not added, glutamate was required by S. mutans GS-5. Aerobic incubations, with carbonate or bicarbonate added, resluted in requirements for glutamate and leucine by several strains. With these incubation conditions, one strain required isoleucine (S. mutans FA-1), another valine (S. mutans AHT), and a third tyrosine (72x46). Aerobic incubations in the absence of carbonate or bicarbonate further increased the number of amino acids required by several strains. Furthermore, when stationary-phase cultures replaced exponentially growing cultures as an inoculum, several strains required additional amino acids, presumably for the initiation of growth.</jats:p

Analysis of growth rate in sucrose-supplemented cultures of Streptococcus mutans

In the presence of sucrose, Streptococcus mutans grows in large glucan-containing aggregates. Because of reports of linear rather than exponential growth of sucrose-grown cultures, the kinetics of growth of sucrose-grown cultures of S. mutans strain OMZ-176 were compared with those of glucose-grown cultures. Culture turbidity measurements indicated that growth of sucrose cultures was slower, did not follow exponential kinetics, and slowed and stopped at lower absorbance values than did glucose-grown cultures. However, measurements of the rates of accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein using fully equilibrated radioactively labeled precursors of each of these macromolecular species in sucrose and glucose-grown cultures showed that: (i) for glucose cultures the synthesis of each of the three informational molecules occurred at the same exponential rate, which was identical to the rate of turbidity increase; (ii) for sucrose cultures each macromolecular species was synthesized at the same exponential rate and these rates were identical to the rate of increase of turbidity of the glucose-grown culture for periods of up to 7 h. Furthermore, the ratios of DNA to RNA, RNA to protein, and protein to DNA for the sucrose cultures were identical to those for the glucose cultures for up to 10 doublings. From these data it was concluded that in the presence of sucrose S. mutans grows in a balanced fashion at the same exponential rate as it does in glucose. The deviation from an exponential growth model of the absorbance in sucrose cultures was attributed to an optical artifact due to the formation of large glucan-containing aggregates of cells. The addition of dextranase to sucrose cultures resulted in cultures which increased in turbidity at the same exponential rate as glucose-grown cultures, without affecting the rate or extent of macromolecular synthesis.</jats:p

Growth of several cariogenic strains of oral streptococci in a chemically defined medium

A chemically defined medium in which Streptococcus mutans strains AHT, BHT, GS-5, JC-2, Ingbritt, At6T, At9T, 6715, and OMZ-176 and Streptococcus salivarius strain HHT grew rapidly to high turbidities was formulated. Maximal turbidities of each strain were observed after 8 to 12 h of aerobic growth. The subsequent transfer of exponentially growing cells into fresh medium resulted in growth at the same rate without lag. Growth of these strains occurred with rates at least one-half of those observed in an organic medium, such as Todd-Hewitt broth. S. mutans strains FA-1 and OMZ-61 grew at relatively slow rates in the defined medium, but more rapidly growth to higher turbidities of both strains was obtained when sodium bicarbonate was added to the medium. Streptococcus sanguis strain OMZ-9 and another group H streptococcus (strain 72 times 46) grew rapidly in the defined medium after the addition of sodium carbonate. The presence of carbonate or bicarbonate yielded higher turbidities of all the other strains, and the growth rates of several of the strains tested were also increased.</jats:p