ABC transporter content diversity inStreptococcus pneumoniaeimpacts competence regulation and bacteriocin production

The opportunistic pathogenStreptococcus pneumoniae(pneumococcus) uses natural genetic competence to increase its adaptability through horizontal gene transfer. One method of acquiring DNA is through predation of neighboring strains with antimicrobial peptides called “bacteriocins.” Competence and production of the major family of pneumococcal bacteriocins, pneumocins, are regulated by the quorum-sensing systemscomandblp, respectively. In the classical paradigm, the ABC transporters ComAB and BlpAB each secretes its own system’s signaling pheromone and in the case of BlpAB also secretes the pneumocins. While ComAB is found in all pneumococci, only 25% of strains encode an intact version of BlpAB [BlpAB(+)] while the rest do not [BlpAB(−)]. Contrary to the classical paradigm, it was previously shown that BlpAB(−) strains can activateblpthrough ComAB-mediated secretion of theblppheromone during brief periods of competence. To better understand the full extent ofcom-blpcrosstalk, we examined the contribution of each transporter to competence development and pneumocin secretion. We found that BlpAB(+) strains have a greater capacity for competence activation through BlpAB-mediated secretion of thecompheromone. Similarly, we show that ComAB and BlpAB are promiscuous and both can secrete pneumocins. Consequently, differences in pneumocin secretion between BlpAB(+) and BlpAB(−) strains derive from the regulation and kinetics of transporter expression rather than substrate specificity. We speculate that BlpAB(−) strains (opportunists) use pneumocins mainly in a narrowly tailored role for DNA acquisition and defense during competence while BlpAB(+) strains (aggressors) expand their use for the general inhibition of rival strains.

Competence Inhibition by the XrpA Peptide Encoded Within thecomXGene ofStreptococcus mutans

SUMMARYStreptococcus mutansdisplays complex regulation of natural genetic competence. Competence development inS. mutansis controlled by a peptide derived from ComS (XIP); which along with the cytosolic regulator ComR controls the expression of the alternative sigma factorcomX, the master regulator of competence development. Recently, a gene embedded within the coding region ofcomXwas discovered and designatedxrpA(comXregulatorypeptideA). XrpA was found to be an antagonist of ComX, but the mechanism was not established. In this study, we reveal through both genomic and proteomic techniques that XrpA is the first describe negative regulator of ComRS systems in streptococci. Transcriptomic and promoter activity assays in the ΔxrpAstrain revealed an up-regulation of genes controlled by both the ComR- and ComX-regulons. Anin vivoprotein crosslinking andin vitrofluorescent polarization assays confirmed that the N-terminal region of XrpA were found to be sufficient in inhibiting ComR-XIP complex binding to ECom-box located within thecomXpromoter. This inhibitory activity was sufficient for decreases in PcomXactivity, transformability and ComX accumulation. XrpA serving as a modulator of ComRS activity ultimately results in changes to subpopulation behaviors and cell fate during competence activation.ABBREVIATED SUMMARYStreptococcus mutansdisplays complex regulation of natural genetic competence, highlighted by a novel gene,xrpA, embedded within the coding region for the master regulator ComX. We show that XrpA modulates ComRS-dependent activation ofcomXexpression, resulting in changes to sub-population behaviors, including cell lysis. XrpA is the first described inhibitor of a ComRS system and, because it is unique toS. mutansit may be targetable to prevent diseases caused by this pathogen.

Natural Competence in Thermoanaerobacter and Thermoanaerobacterium Species

ABSTRACT Low-G+C thermophilic obligate anaerobes in the class Clostridia are considered among the bacteria most resistant to genetic engineering due to the difficulty of introducing foreign DNA, thus limiting the ability to study and exploit their native hydrolytic and fermentative capabilities. Here, we report evidence of natural genetic competence in 13 Thermoanaerobacter and Thermoanaerobacterium strains previously believed to be difficult to transform or genetically recalcitrant. In Thermoanaerobacterium saccharolyticum JW/SL-YS485, natural competence-mediated DNA incorporation occurs during the exponential growth phase with both replicating plasmid and homologous recombination-based integration, and circular or linear DNA. In T. saccharolyticum, disruptions of genes similar to comEA, comEC, and a type IV pilus (T4P) gene operon result in strains unable to incorporate further DNA, suggesting that natural competence occurs via a conserved Gram-positive mechanism. The relative ease of employing natural competence for gene transfer should foster genetic engineering in these industrially relevant organisms, and understanding the mechanisms underlying natural competence may be useful in increasing the applicability of genetic tools to difficult-to-transform organisms.

DNA as a Nutrient: Novel Role for Bacterial Competence Gene Homologs

ABSTRACT The uptake and stable maintenance of extracellular DNA, genetic transformation, is universally recognized as a major force in microbial evolution. We show here that extracellular DNA, both homospecific and heterospecific, can also serve as the sole source of carbon and energy supporting microbial growth. Mutants unable to consume DNA suffer a significant loss of fitness during stationary-phase competition. InEscherichia coli, the use of DNA as a nutrient depends on homologs of proteins involved in natural genetic competence and transformation in Haemophilus influenzae andNeisseria gonorrhoeae. Homologs of these E. coli genes are present in many members of the γ subclass ofProteobacteria, suggesting that the mechanisms for consumption of DNA may have been widely conserved during evolution.

Natural Genetic Competence in Bacillus subtilis Natto OK2

ABSTRACT We isolated a Bacillus subtilis natto strain, designated OK2, from a lot of commercial fermented soybean natto and studied its ability to undergo natural competence development using acomG-lacZ fusion at the amyE locus. Although transcription of the late competence genes was not detected in theB. subtilis natto strain OK2 during competence development, these genes were constitutively transcribed in the OK2 strain carrying either the mecA or the clpC mutation derived from B. subtilis 168. In addition, both OK2 mutants exhibited high transformation frequencies, comparable with that observed for B. subtilis 168. Moreover, as expected from these results, overproduction of ComK derived from strain 168 in strain OK2 resulted in a high transformation frequency as well as in induction of the late competence genes. These results clearly indicated that ComK produced in both the mecA and clpC mutants of strain OK2 (ComKOK2) could activate the transcription of the whole set of late competence genes and suggested that ComKOK2 was not activated in strain OK2 during competence development. We therefore sequenced the comS gene of OK2 and compared it with that of 168. The comS OK2had a single-base change, resulting in the replacement of Ser (strain 168) by Cys (strain OK2) at position 11.