Impact of Orthologous Gene Replacement on the Circuitry Governing Pilus Gene Transcription in Streptococci

The evolutionary history of several genes of the bacterial pathogen Streptococcus pyogenes strongly suggests an origin in another species, acquired via replacement of the counterpart gene (ortholog) following a recombination event. An example of orthologous gene replacement is provided by the nra/rofA locus, which encodes a key regulator of pilus gene transcription. Of biological importance is the previous finding that the presence of the nra- and rofA-lineage alleles, which are approximately 35% divergent, correlates strongly with genetic markers for streptococcal infection at different tissue sites in the human host (skin, throat).In this report, the impact of orthologous gene replacement targeting the nra/rofA locus is experimentally addressed. Replacement of the native nra-lineage allele with a rofA-lineage allele, plus their respective upstream regions, preserved the polarity of Nra effects on pilus gene transcription (i.e., activation) in the skin strain Alab49. Increased pilus gene transcription in the rofA chimera correlated with a higher rate of bacterial growth at the skin. The transcriptional regulator MsmR, which represses nra and pilus gene transcription in the Alab49 parent strain, has a slight activating effect on pilus gene expression in the rofA chimera construct.Data show that exchange of orthologous forms of a regulatory gene is stable and robust, and pathogenicity is preserved. Yet, new phenotypes may also be introduced by altering the circuitry within a complex transcriptional regulatory network. It is proposed that orthologous gene replacement via interspecies exchange is an important mechanism in the evolution of highly recombining bacteria such as S. pyogenes

CbgA, a Protein Involved in Cortex Formation and Stress Resistance in Myxococcus xanthus Spores

CbgA plays a role in cortex formation and the acquisition of a subset of stress resistance properties in Myxococcus xanthus spores. The cbgA mutant produces spores with thin or no cortex layers, and these spores are more sensitive to heat and sodium dodecyl sulfate than their wild-type counterparts

Identification of Major Sporulation Proteins of Myxococcus xanthus Using a Proteomic Approach

Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress

Spaceflight promotes biofilm formation by Pseudomonas aeruginosa.

Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight

Spaceflight increases biofilm formation by <i>P. aeruginosa</i>.

<p>Wild-type <i>P. aeruginosa</i> was cultured under normal gravity (black bars) and spaceflight (grey bars) conditions in mAUM or mAUMg containing 5 or 50 mM phosphate. (<b>A</b>) The number of surface-associated viable cells per cellulose ester membrane. (<b>B</b>) Biofilm biomass and (<b>C</b>) mean biofilm thickness were quantified by analysis of CLSM images. Error bars, SD; N = 3. *<i>p≤0.05</i>, **<i>p</i>≤<i>0.01</i>.</p

Illustration summarizing the influence of gravity, flow, and motility on <i>P.aeruginosa</i> biofilm architecture.

<p>Illustration summarizing the influence of gravity, flow, and motility on <i>P.aeruginosa</i> biofilm architecture.</p

Spaceflight and motility affect biofilm formation and architecture.

<p>Wild type, <i>ΔmotABCD,</i> and <i>ΔpilB</i> were grown in mAUMg with solid inserts or GE inserts. Biomass and mean thickness were calculated from CLSM images using COMSTAT. Results are shown as mean ± SD; N = 3. ND, not determined.</p

<i>P.aeruginosa</i> biofilms cultured during spaceflight display column-and-canopy structures.

<p>Confocal laser scanning micrographs of 3-day-old biofilms formed by wild type, <i>ΔmotABCD</i>, and <i>ΔpilB</i> comparing normal gravity and spaceflight culture conditions. All strains were grown in mAUMg with 5 mM phosphate. No significant differences in structure or thickness were observed with mAUMg containing 5 or 50 mM phosphate. (<b>A</b>) Representative side-view images. (<b>B</b>) Representative 5.8 µm thick slices generated from partial <i>z</i> stacks. Maximum thickness is indicated in the upper right corner of the top slice for each condition.</p

Increased oxygen availability minimizes gravitational effects on biofilm formation by <i>P.aeruginosa</i>.

<p>Representative side view confocal laser scanning micrographs of 3-day-old biofilms formed by wild-type <i>P. aeruginosa</i> and <i>ΔmotABCD</i> grown in mAUMg with gas exchange (GE) inserts comparing normal gravity and spaceflight culture conditions.</p