Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression

The P_R promoter of mycobacteriophage BPs directs early lytic gene expression and is under the control of the BPs repressor, gp33. Reporter gene fusions showed that P_R has modest activity in an extrachromosomal context but has activity that is barely detectable in an integrated context, even in the absence of its repressor. Mutational dissection of P_R showed that it uses a canonical −10 hexamer recognized by SigA, and mutants with mutations to the sequence 5′-TATAMT had the greatest activities. It does not contain a 5′-TGN-extended −10 sequence, although mutants with mutations creating an extended −10 sequence had substantially increased promoter activity. Mutations in the −35 hexamer also influenced promoter activity but were strongly context dependent, and similar substitutions in the −35 hexamer differentially affected promoter activity, depending on the −10 and extended −10 motifs. This warrants caution in the construction of synthetic promoters or the bioinformatic prediction of promoter activity. Combinations of mutations throughout P_R generated a calibrated series of promoters for expression of stably integrated recombinant genes in both Mycobacterium smegmatis and M. tuberculosis, with maximal promoter activity being more than 2-fold that of the strong hsp60 promoter.     

Repressed Quorum sensing by overexpressing LsrR Hampers Salmonella evasion from oxidative killing within macrophages

Bacterial cell-to-cell communication, termed quorum sensing (QS), leads to coordinated group behavior in a cell-density-dependent fashion and controls a variety of physiological processes including virulence gene expression. The repressor of the lsr operon, LsrR, is the only known regulator of LuxS/AI-2-mediated QS in Salmonella. Although lack of lsrR did not result in noticeable differences in Salmonella survival, the down-regulation of QS as a result of lsrR overexpression decreased Salmonella survival within macrophages. We found that impaired growth of Salmonella overexpressing lsrR within macrophages was due largely to its hypersensitivity to NADPH-dependent oxidative stress. This, in turn, was a result of decreased expression of genes involved in the oxidative stress response, such as sodA, sodCI, and sodCII, when lsrR was overexpressed. These results suggest that down-regulation of QS by excess LsrR can lower Salmonella virulence by hampering Salmonella evasion from oxidative killing within macrophages.     

Quorum sensing provides a molecular mechanism for evolution to tune and maintain investment in cooperation

As selection frequently favors noncooperating defectors in mixed populations with cooperators, mechanisms that promote cooperation stability clearly exist. One potential mechanism is bacterial cell-to-cell communication, quorum sensing (QS), which can allow cooperators to prevent invasion by defectors. However, the impact of QS on widespread maintenance of cooperation in well-mixed conditions has not been experimentally demonstrated over extended evolutionary timescales. Here, we use wild-type (WT) Vibrio campbellii that regulates cooperation with QS and an unconditional cooperating (UC) mutant to examine the evolutionary origins and dynamics of novel defectors during a long-term evolution experiment. We found that UC lineages were completely outcompeted by defectors, whereas functioning QS enabled the maintenance of cooperative variants in most WT populations. Sequencing evolved populations revealed multiple luxR mutations that swept the UC lineages. However, the evolution of mutant lineages with reduced levels of bioluminescence (dims) occurred in many WT lineages. These dim variants also decreased other cooperative phenotypes regulated by QS, including protease production, indicating they result from changes to QS regulation. This diminished investment phenotype optimizes a tradeoff between cooperative input and growth output and suggests that decreasing the cost of QS could be a favorable strategy for maintaining the cooperative behaviors it regulates.Subject terms: Bacterial evolution, Microbial ecology, Molecular evolution     

Recognition sequences of repressor and polymerase in the operators of bacteriophage lambda

Nucleotide sequences in two wild-type and six mutant operators in the DNA of phage λ are compared. Strikingly similar 17 base pair units are found which we identify as the repressor binding sites. Each operator contains multiple repressor binding sites separated by A-T rich spacers. Elements of 2 fold rotational symmetry are present in each of the sites. Superimposed on each operator is an E. coli RNA polymerase recognition site (promoter). Similarities in the sequences of the two λ promoters, a lac promoter, and an E. coli RNA polymerase recognition site in SV40 DNA are noted.     

Chemical synthesis and biochemical reactivity of bacteriophage lambda PR promoter

By a combination of chemical and enzymatic methods, a 75 base pair DNA duplex containing the sequence of the lambda PR promoter including the OR1 and OR2 cI repressor binding sites was synthesized. The solid support phosphite triester procedure (Caruthers, M. H. et al., Cold Spring Harbor Symposia on Quantitative Biology XLVII, in press) was used for the synthesis of oligonucleotides comprising the sequence. We report here an adaptation of the method of DNA synthesis in test tubes. Assembly of the oligonucleotides involved the use of T4 polynucleotide kinase and T4 DNA ligase. We show that the synthetic DNA is recognized by RNA polymerase and cI repressor in a manner identical to the same control region contained on a restriction fragment isolated from bacteriophage lambda DNA. Our synthetic approach using chemically synthesized promoter variants is thus suitable for studies probing the function of promoters.     

Synthetic Promoters: Designing the <Emphasis Type="Italic">cis</Emphasis> Regulatory Modules for Controlled Gene Expression

Designing the expression cassettes with desired properties remains the most important consideration of gene engineering technology. One of the challenges for predictive gene expression is the modeling of synthetic gene switches to regulate one or more target genes which would directly respond to specific chemical, environmental, and physiological stimuli. Assessment of natural promoter, high-throughput sequencing, and modern biotech inventory aided in deciphering the structure of cis elements and molding the native cis elements into desired synthetic promoter. Synthetic promoters which are molded by rearrangement of cis motifs can greatly benefit plant biotechnology applications. This review gives a glimpse of the manual in vivo gene regulation through synthetic promoters. It summarizes the integrative design strategy of synthetic promoters and enumerates five approaches for constructing synthetic promoters. Insights into the pattern of cis regulatory elements in the pursuit of desirable “gene switches” to date has also been reevaluated. Joint strategies of bioinformatics modeling and randomized biochemical synthesis are addressed in an effort to construct synthetic promoters for intricate gene regulation.