No abstract

Maintaining the integrity of the genome is essential to cell survival. In the bacterium Caulobacter crescentus, the single circular chromosome exhibits a specific orientation in the cell, with the replication origin (ori) residing at the pole of the cell bearing a stalk. Upon initiation of replication, the duplicated centromere-like region parS and ori move rapidly to the opposite pole where parS is captured by a microdomain hosting a unique set of proteins that contribute to the identity of progeny cells. Many questions remain as to how this organization is maintained. In this study, we constructed strains of Caulobacter in which ori and the parS centromere can be induced to move to the opposite cell pole in the absence of chromosome replication, allowing us to ask whether once these chromosomal foci were positioned at the wrong pole, replication initiation and chromosome segregation can proceed in the opposite orientation. Our data reveal that DnaA can initiate replication and ParA can orchestrate segregation from either cell pole. The cell reconstructs the organization of its ParA gradient in the opposite orientation to segregate one replicated centromere from the new pole toward the stalked pole (i.e., opposite direction), while displaying no detectable viability defects. Thus, the unique polar microdomains exhibit remarkable flexibility in serving as a platform for directional chromosome segregation along the long axis of the cell. IMPORTANCE Bacteria can accomplish surprising levels of organization in the absence of membrane organelles by constructing subcellular asymmetric protein gradients. These gradients are composed of regulators that can either trigger or inhibit cell cycle events from distinct cell poles. In Caulobacter crescentus, the onset of chromosome replication and segregation from the stalked pole are regulated by asymmetric protein gradients. We show that the activators of chromosome replication and segregation are not restricted to the stalked pole and that their organization and directionality can be flipped in orientation. Our results also indicate that the subcellular location of key chromosomal loci play important roles in the establishment of the asymmetric organization of cell cycle regulators.

The accurate onset of chromosome replication and segregation are fundamental for the survival of the cell. In bacteria, regulation of chromosome replication lies primarily at the initiation step. The bacterial replication initiator DnaA recognizes the origin of replication (ori) and opens this double stranded site allowing for the assembly of the DNA replication machinery. Following the onset of replication initiation, the partitioning protein ParA triggers the onset of chromosome segregation by direct interactions with ParB-bound to the centromere. The subcellular organization of ori and centromere are maintained after the completion of each cell cycle. It remains unclear what triggers the onset of these key chromosome regulators DnaA and ParA. One potential scenario is that the microenvironment of where the onset of replication and segregation take place hosts the regulators that trigger the activity of DnaA and ParA. In order to address this, we analyzed whether the activity of DnaA and ParA are restricted to only one site within the cell. In non-dividing cells of the alpha proteobacterium Caulobacter crescentus, ori and centromere are found near the stalked pole. To test DnaA's ability to initiate replication away from the stalked pole, we engineered a strain where movement of ori was induced in the absence of chromosome replication. Our data show that DnaA can initiate replication of the chromosome independently of the subcellular localization of ori. Furthermore, we discovered that the partitioning protein ParA was functional and could segregate the replicated centromere in the opposite direction from the new pole toward the stalked pole. We showed that the organization of the ParA gradient can be completely reconstructed in the opposite orientation by rearranging the location of the centromere. Our data reveal the high flexibility of the machineries that trigger the onset of chromosome replication and segregation in bacteria. Our work also provides insights into the coordination between replication and segregation with the cellular organization of specific chromosomal loci.

Bacterial ATP binding cassette (ABC) transporters mediate the influx of numerous substrates. The cluster A-I ABC transporters are responsible for the specific uptake of the essential metals zinc, manganese or iron, making them necessary for survival in metal-limited environments, which for pathogens include the animal host. In Paracoccus denitrificans, there are two zinc ABC transporter systems: ZnuABC and AztABCD with apparently redundant functions under zinc-limited conditions. The unusual presence of two zinc ABC transporter systems in the same organism allowed for the investigation of specificity in the interaction between the solute binding protein (SBP) and its cognate permease. We also assessed the role of flexible loop features in the SBP in permease binding and zinc transport. The results indicate that the SBP–permease interaction is highly specific and does not require the flexible loop features of the SBP. We also present an expanded table of the properties of characterized cluster A-I SBPs and a multiple sequence alignment highlighting the conserved features. Through this analysis, an apparently new family of binding proteins associated with ABC transporters was identified. The presence of homologues in several human pathogens raises the possibility of using it as a target for the development of new antimicrobial therapies.

ATP binding cassette (ABC) transporters are the primary means by which bacteria acquire trace elements from the environment. They rely on solute binding proteins (SBPs) to bind the relevant substrate and deliver it to the integral membrane permease for ATP-powered import into the cytoplasm. SBPs of cluster A-I are known to facilitate the transport of essential metals zinc, manganese, and iron, and many have been characterized to date. A group of ABC transporter operons dubbed zinc-regulated genes (zrg) have recently been shown to transport zinc with putative SBPs (zrgA) bearing no homology to the classical cluster A-I family, and a recent crystal structure of a representative protein from Pseudomonas aeruginosa shows no structural similarity to classical SBPs. Thus, the ZrgA proteins appear to represent a newly discovered family of zinc SBPs widespread among Gram-negative bacteria, including human pathogens. Here, we have determined the crystal structure of ZrgA from Vibrio cholerae and characterized its zinc binding in vitro and function in vivo. We also assessed the role of a histidine-rich sequence that appears to be a hallmark of ZrgA proteins that is particularly long in V. cholerae ZrgA. The results show that the zrgA gene is critical to the function of the operon, consistent with a function as an SBP in this system. Further, the His-rich region is not essential to the function of ZrgA, but it does provide additional zinc binding sites in vitro. The structure and zinc binding data for ZrgA reveal interesting differences between it and its homologue from P. aeruginosa, illustrating diversity within this little-studied protein family.