In recent years, there has been an increasing interest in the exploitation of microalgae in industrial biotechnology. Potentially, these phototrophic eukaryotes could be used for the low-cost synthesis of valuable recombinant products such as bioactive metabolites and therapeutic proteins. The algal chloroplast in particular represents an attractive target for such genetic engineering, both because it houses major metabolic pathways and because foreign genes can be targeted to specific loci within the chloroplast genome, resulting in high-level, stable expression. However, routine methods for chloroplast genetic engineering are currently available only for one species—Chlamydomonas reinhardtii—and even here, there are limitations to the existing technology, including the need for an expensive biolistic device for DNA delivery, the lack of robust expression vectors, and the undesirable use of antibiotic resistance markers. Here, we describe a new strain and vectors for targeted insertion of transgenes into a neutral chloroplast locus that (i) allow scar-less fusion of a transgenic coding sequence to the promoter/5′UTR element of the highly expressed endogenous genes psaA or atpA, (ii) employ the endogenous gene psbH as an effective but benign selectable marker, and (iii) ensure the successful integration of the transgene construct in all transformant lines. Transformation is achieved by a simple and cheap method of agitation of a DNA/cell suspension with glass beads, with selection based on the phototrophic rescue of a cell wall-deficient ΔpsbH strain. We demonstrate the utility of these tools in the creation of a transgenic line that produces high levels of functional human growth hormone.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7354-6) contains supplementary material, which is available to authorized users.

A range of properties, including the ability to utilize various sugars, bind macromolecules and produce mutacins, are known to vary in their occurrence in different strains of Streptococcus mutans. In addition, insertion-sequence elements show a limited distribution and sequencing of the genome of S. mutans UA159 has revealed the presence of putative genomic islands of atypical base composition indicative of foreign DNA. PCR primers flanking regions suspected of having inserted DNA were designed on the basis of the genome sequence of S. mutans UA159 and used to explore variation in a collection of 39 strains isolated in various parts of the world over the last 40 years. Extensive differences between strains were detected, and similar insertion/deletion events appear to be present in the genomes of strains with very different origins. In two instances, insertion of foreign DNA appears to have displaced original S. mutans genes. Together with previous results on the occurrence of deletions in genes associated with sugar metabolism, the results indicate that S. mutans has a core genome and a dispensable genome, and that dispensable genes have become widely distributed through horizontal transfer.

The concept of an infectious agent playing a role in cardiovascular disease is slowly gaining attention. Among several pathogens identified, the oral bacterium Streptococcus gordonii has been implicated as a plausible agent. Platelet adhesion and subsequent aggregation are critical events in the pathogenesis and dissemination of the infective process. Here we describe the identification and characterization of a novel cell wall-anchored surface protein, PadA (397 kDa), of S. gordonii DL1 that binds to the platelet fibrinogen receptor GPIIbIIIa. Wild-type S. gordonii cells induced platelet aggregation and supported platelet adhesion in a GPIIbIIIa-dependent manner. Deletion of the padA gene had no effect on platelet aggregation by S. gordonii but significantly reduced (>75%) platelet adhesion to S. gordonii. Purified N-terminal PadA recombinant polypeptide adhered to platelets. The padA mutant was unaffected in production of other platelet-interactive surface proteins (Hsa, SspA, and SspB), and levels of adherence of the mutant to fetuin or platelet receptor GPIb were unaffected. Wild-type S. gordonii, but not the padA mutant, bound to Chinese hamster ovary cells stably transfected with GPIIbIIIa, and this interaction was ablated by addition of GPIIbIIIa inhibitor Abciximab. These results highlight the growing complexity of interactions between S. gordonii and platelets and demonstrate a new mechanism by which the bacterium could contribute to unwanted thrombosis.

The basis for genotypic and phenotypic variation within Streptococcus mutans is poorly understood but the availability of the genome sequence of strain UA159 provides an opportunity for comparative studies. Genomic DNA prepared from nine strains of S. mutans was used to probe a microarray consisting of oligonucleotides representing 1948 open reading frames of S. mutans UA159. A total of 385 (20%) of the UA159 open reading frames were found to be absent from one or more of the test strains. Absent open reading frames frequently occurred in blocks of adjacent open reading frames and represented regions previously experimentally detected by polymerase chain reaction, predicted genomic islands and insertion sequence elements as well as novel open reading frames. Approximately half appear to involve foreign DNA acquired by horizontal transmission. The results indicate the existence of distinct core and dispensable genomes and may help explain the phenotypic and genotypic variation within S. mutans.