The commitment of differentiating cells to a specialized fate is fundamental to the correct assembly of tissues within a multicellular organism. Because commitment is often irreversible, entry into and progression through this phase of development must be tightly regulated. Under nitrogen-limiting conditions, the multicellular cyanobacterium Anabaena sp. strain PCC 7120 terminally commits ∼10% of its cells to become specialized nitrogen-fixing heterocysts. Although commitment is known to occur 9–14 h after the induction of differentiation, the factors that regulate the initiation and duration of this phase have yet to be elucidated. Here, we report the identification of four genes that share a functional domain and modulate heterocyst commitment: hetP (alr2818), asl1930, alr2902, and alr3234. Epistatic relationships between all four genes relating to commitment were revealed by deleting them individually and in combination; asl1930 and alr3234 acted most upstream to delay commitment, alr2902 acted next in the pathway to inhibit development, and hetP acted most downstream to drive commitment forward. Possible protein–protein interactions between HetP, its homologs, and the heterocyst master regulator, HetR, were assessed, and interaction partners were defined. Finally, patterns of gene expression for each homolog, as determined by promoter fusions to gfp and reverse transcription–quantitative PCR, were distinct from that of hetP in both spatiotemporal organization and regulation. We posit that a dynamic succession of protein–protein interactions modulates the timing and efficiency of the commitment phase of development and note that this work highlights the utility of a multicellular cyanobacterium as a model for the study of developmental processes.

Chlamydia trachomatis infections are the leading cause of sexually transmitted infections of bacterial origin. Lower genital tract infections are often asymptomatic, and therefore left untreated, leading to ascending infections that have long-term consequences on female reproductive health. Human pathology can be recapitulated in mice with the mouse adapted strain C . muridarum . Eight years into the post-genetic era, significant advances to expand the Chlamydia genetic toolbox have been made to facilitate the study of this important human pathogen. However, the need for additional tools remains, especially for C . muridarum . Here, we describe a new set of spectinomycin resistant E . coli-Chlamydia shuttle vectors, for C . trachomatis and C . muridarum . These versatile vectors allow for expression and localization studies of Chlamydia effectors, such as Inc proteins, and will be instrumental for mutant complementation studies. In addition, we have exploited the differential expression of specific Chlamydia genes during the developmental cycle to engineer an omcA :: gfp fluorescent transcriptional reporter. This novel tool allows for monitoring RB to EB conversion at the bacterial level. Spatiotemporal tracking of GFP expression within individual inclusions revealed that RB to EB conversion initiates in bacteria located at the edge of the inclusion and correlates with the time post initiation of bacterial replication and inclusion size. Comparison between primary and secondary inclusions potentially suggests that the environment in which the inclusions develop influences the timing of conversion. Altogether, the Chlamydia genetic tools described here will benefit the field, as we continue to investigate the molecular mechanisms underlying Chlamydia -host interaction and pathogenesis.

Membrane contact sites (MCS) are crucial for nonvesicular trafficking-based interorganelle communication. Endoplasmic reticulum (ER)–organelle tethering occurs in part through the interaction of the ER resident protein VAP with FFAT motif-containing proteins. FFAT motifs are characterized by a seven amino acidic core surrounded by acid tracks. We have previously shown that the human intracellular bacterial pathogen Chlamydia trachomatis establishes MCS between its vacuole (the inclusion) and the ER through expression of a bacterial tether, IncV, displaying molecular mimicry of eukaryotic FFAT motif cores. Here, we show that multiple layers of host cell kinase-mediated phosphorylation events govern the assembly of the IncV–VAP tethering complex and the formation of ER-Inclusion MCS. Via a C-terminal region containing three CK2 phosphorylation motifs, IncV recruits CK2 to the inclusion leading to IncV hyperphosphorylation of the noncanonical FFAT motif core and serine-rich tracts immediately upstream of IncV FFAT motif cores. Phosphorylatable serine tracts, rather than genetically encoded acidic tracts, accommodate Type III-mediated translocation of IncV to the inclusion membrane, while achieving full mimicry of FFAT motifs. Thus, regulatory components and post-translational modifications are integral to MCS biology, and intracellular pathogens such as C. trachomatis have evolved complex molecular mimicry of these eukaryotic features.

The obligate intracellular pathogen Chlamydia trachomatis is the leading cause of non-congenital blindness and causative agent of the most common sexually transmitted infection of bacterial origin. With a reduced genome, C. trachomatis is dependent on its host for survival, in part due to a need for the host cell to compensate for incomplete bacterial metabolic pathways. However, relatively little is known regarding how C. trachomatis is able to hijack host cell metabolism. In this study, we show that two host glycolytic enzymes, Aldolase A and Pyruvate Kinase, as well as Lactate Dehydrogenase, are enriched at the C. trachomatis inclusion membrane during infection. Inclusion localization was not species specific, as a similar phenotype was observed with C. muridarum. Time course experiments showed that the number of positive inclusions increased throughout the developmental cycle. Additionally, these host enzymes co-localized to the same inclusion and their localization did not appear dependent on sustained bacterial protein synthesis, or intact host actin, vesicular trafficking, or microtubules. Depletion of the host glycolytic enzyme Aldolase A resulted in decreased inclusion size and infectious progeny production, indicating a role for host glycolysis in bacterial growth. Finally, quantitative PCR analysis showed that expression of C. trachomatis glycolytic enzymes inversely correlated with host enzymes localization at the inclusion. We discuss potential mechanisms leading to inclusion localization of host glycolytic enzymes and how it could benefit the bacteria. Altogether, our findings provide further insight into the intricate relationship between host and bacterial metabolism during Chlamydia infection.

No abstract