We performed bottom-up engineering of a synthetic pathway in Escherichia coli for the production of eukaryotic trimannosyl chitobiose glycans and the transfer of these glycans to specific asparagine residues in target proteins. The glycan biosynthesis was enabled by four eukaryotic glycosyltransferases, including the yeast uridine diphosphate-N-acetylglucosamine transferases Alg13 and Alg14 and the mannosyltransferases Alg1 and Alg2. By including the bacterial oligosaccharyltransferase PglB from Campylobacter jejuni, we successfully transferred glycans to eukaryotic proteins.

Streptococcus mutans is generally recognized as a causative agent of human dental caries. The production of mutacins (bacteriocins) by S. mutans is considered to be an important factor in the colonization and establishment of S. mutans in the dental biofilm. Two types of mutacins have been characterized: the lantibiotics and the non-lantibiotics. The lantibiotics generally have a wider spectrum of activity than the non-lantibiotics, which make them attractive targets for development into new antimicrobial modalities. The non-lantibiotics are much more prevalent among strains of S. mutans and play a significant role in both community-level and population-level interactions in the dental biofilm. These interactions are directly mediated through the ComCDE two-component system and the newly characterized LytTR Regulation Systems HdrRM and BrsRM. These systems coordinate natural competence development and mutacin production as a means to acquire transforming DNA either by killing closely related streptococcal species in the vicinity of S. mutans, or through an altruistic suicide mechanism among a subpopulation of competent cells within the S. mutans community. As more S. mutans strains are sequenced, it is anticipated that additional mutacins with novel functions will be discovered, which may yield further insights into the ecological role of mutacins within the oral biofilm.

We have constructed the first Escherichia coli-Veillonella shuttle vector based on an endogenous plasmid (pVJL1) isolated from a clinical Veillonella strain. A highly transformable Veillonella strain was also identified. Both the shuttle vector and the transformable strain should be valuable tools for future Veillonella genetic studies.

Certain oral streptococci produce H(2)O(2) under aerobic growth conditions to inhibit competing species like Streptococcus mutans. Additionally, H(2)O(2) production causes the release of extracellular DNA (eDNA). eDNA can serve in several important functions: biofilm formation and cell-cell aggregation is supported by eDNA, while eDNA can serve as a nutrient and has antimicrobial properties by chelating essential cations. Since eDNA contains larger fragments of DNA, it has the potential to transfer genomic information. Using Streptococcus gordonii as a model organism for streptococcal H(2)O(2) production, H(2)O(2) dependent eDNA release was further investigated. Using defined growth conditions, the eDNA release process was shown to be entirely dependent on H(2)O(2). Chromosomal DNA damage seems to be the intrinsic signal for the release. Though, only actively growing cells were proficient eDNA donors. Interestingly, the process of eDNA production was found to be coupled with the induction of the S. gordonii natural competence system. Consequently, the production of H(2)O(2) triggered the transfer of antibiotic resistance genes. These results suggest that H(2)O(2) production is potentially much more than a simple toxic metabolic byproduct, rather it could serve as an important environmental signal that facilitates species evolution by transfer of genetic information and an increase in the mutation rate.

Pseudomonas aeruginosa is an opportunistic pathogen capable of group behaviors including biofilm formation and swarming motility. These group behaviors are regulated by both the intracellular signaling molecule c-di-GMP and acylhomoserine lactone quorum sensing systems. Here, we show that the Pseudomonas quinolone signal (PQS) system also contributes to the regulation of swarming motility. Specifically, our data indicate that HHQ, a precursor of PQS, likely induces the production of the phenazine PCA, which in turn acts via an as-yet unknown downstream mechanism to repress swarming motility. We show that this HHQ- and PCA-dependent swarming repression is apparently independent of changes in global levels of c-di-GMP, suggesting complex regulation of this group behavior.

Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed, which utilize either temperature sensitive plasmids or counterselectable suicide vectors containing both positive and negative selection markers. For many species, these techniques are not especially useful due to difficulties with cloning in E. coli and/or a lack of functional negative selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable for a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows for extremely quick assembly and eliminates the requirement for either temperature sensitive replicons or suicide vectors. As a proof of principle, we used the Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild type S. mutans strains, we further demonstrate that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches.

Veillonellae are one of the most prevalent and predominant microorganisms in both the supra- and subgingival plaques of the human oral cavity. Veillonellae's mutualistic relationships with the early, middle, and late colonizers of the oral cavity make them an important component of oral biofilm ecology. Unlike other ubiquitous early colonizers in the oral cavity, surprisingly little is known about Veillonella biology due to our lack of ability to genetically transform this group of bacteria. The objective of this study was to test the transformability of veillonellae. Using Veillonella parvula strain PK1910, we first obtained spontaneous mutations conferring streptomycin resistance. These mutations all carry a K43N substitution in the RpsL protein. Using the mutated rpsL gene as a selection marker, a variety of conditions were tested and optimized for electroporation. With the optimized protocol, we were able to introduce the first targeted mutation into the chromosome of V. parvula PK1910. Although more studies are needed to develop a robust genetic manipulation system in veillonellae, our results demonstrated, for the first time, that V. parvula is transformable, at least for strain PK1910.

OBJECTIVES: To compare the effectiveness of bacterial interference versus placebo in preventing urinary tract infection (UTI). METHODS: The main outcome measure was the numbers of episodes of UTI/patient-year. Randomization was computer generated, with allocation concealment by visibly indistinguishable products distributed from a core facility. The healthcare providers and those assessing the outcomes were unaware of the group allocation. Adult patients (n = 65) with neurogenic bladder after spinal cord injury and a history of recurrent UTI were randomized in a 3:1 ratio to receive either Escherichia coli HU2117 or sterile saline. Urine cultures were obtained weekly during the first month and then monthly for 1 year. The patients were evaluable if they remained colonized with E. coli HU2117 for >4 weeks (experimental group). The trial is closed to follow-up. RESULTS: Of the 59 patients who received bladder inoculations, 27 were evaluable (17 in the experimental group and 10 in the placebo group). The 2 study groups had comparable clinical characteristics. Of 17 patients colonized with E. coli HU2117 and the 10 control patients, 5 (29%, 95% confidence interval 0.11-0.56) and 7 (70%, 95% confidence interval 0.35-0.92) developed >1 episode of UTI (P =.049; 1-sided Fisher's exact test), respectively. The average number of episodes of UTI/patient-year was also lower (P =.02, Wilcoxon rank sum test) in the experimental (0.50) than in the control group (1.68). E. coli HU2117 did not cause symptomatic UTI. CONCLUSIONS: Bladder colonization with E. coli HU2117 safely reduces the risk of symptomatic UTI in patients with spinal cord injury. Effective, but less complex, methods for achieving bladder colonization with E. coli HU2117 are under investigation.

In the oral biofilm, the 'mitis' streptococci are among the first group of organisms to colonize the tooth surface. Their proliferation is thought to be an important factor required for antagonizing the growth of cariogenic species such as Streptococcus mutans. In this study, we used a three-species mixed culture to demonstrate that another ubiquitous early colonizing species, Veillonella parvula, can greatly affect the outcome of the competition between a pair of antagonists such as S. mutans and Streptococcus gordonii. Transcriptome analysis further revealed that S. mutans responds differentially to its friend (V. parvula) and foe (S. gordonii). In the mixed culture with S. gordonii, all but one of the S. mutans sugar uptake and metabolic genes were downregulated, while genes for alternative energy source utilization and H₂O₂ tolerance were upregulated, resulting in a slower but persistent growth. In contrast, when cultured with V. parvula, S. mutans grew equally well or better than in monoculture and exhibited relatively few changes within its transcriptome. When V. parvula was introduced into the mixed culture of S. mutans and S. gordonii, it rescued the growth inhibition of S. mutans. In this three-species environment, S. mutans increased the expression of genes required for the uptake and metabolism of minor sugars, while genes required for oxidative stress tolerance were downregulated. We conclude that the major factors that affect the competition between S. mutans and S. gordonii are carbohydrate utilization and H₂O₂ resistance. The presence of V. parvula in the tri-species culture mitigates these two major factors and allows S. mutans to proliferate, despite the presence of S. gordonii.

To colonize the cystic fibrosis lung, Pseudomonas aeruginosa establishes sessile communities referred to as biofilms. Although the signaling molecule c-di-GMP governs the transition from motile to sessile growth, the environmental signal(s) required to modulate biofilm formation remain unclear. Using relevant in vivo concentrations of the 19 amino acids previously identified in cystic fibrosis sputum, we demonstrated that arginine, ornithine, isoleucine, leucine, valine, phenylalanine and tyrosine robustly promoted biofilm formation in vitro. Among the seven biofilm-promoting amino acids, only arginine also completely repressed the ability of P. aeruginosa to swarm over semi-solid surfaces, suggesting that arginine may be an environmental cue favoring a sessile lifestyle. Mutating two documented diguanylate cyclases required for biofilm formation (SadC and RoeA) reduced biofilm formation and restored swarming motility on arginine-containing medium. Growth on arginine increased the intracellular levels of c-di-GMP, and this increase was dependent on the SadC and RoeA diguanylate cyclases. Strains mutated in sadC, roeA or both also showed a reduction in biofilm formation when grown with the other biofilm-promoting amino acids. Taken together, these results suggest that amino acids can modulate biofilm formation and swarming motility, at least in part, by controlling the intracellular levels of c-di-GMP.