The alphaproteobacterium Hyphomonas neptunium proliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk-like extension emanating from the mother cell body. Studies of this species so far have been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies two H. neptunium promoters that are activated specifically by copper and zinc. Functional analyses show that they have low basal activity and a high dynamic range, meeting the requirements for use as a multipurpose expression system. To facilitate their application, the two promoters were incorporated into a set of integrative plasmids, featuring a choice of two different selection markers and various fluorescent protein genes. These constructs enable the straightforward generation and heavy metal-inducible synthesis of fluorescent protein fusions in H. neptunium, thereby opening the door to an in-depth analysis of polar growth and development in this species. Bacteria are a phylogenetically diverse group of organisms whose study has provided important insights into the mechanisms that mediate the spatiotemporal organization of cells. However, most of our knowledge on bacterial cell biology so far has come from the analysis of only a few well-established model species, such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, which typically exhibit a rod-like morphology and divide by symmetric or asymmetric binary fission.To further our understanding of subcellular organization in bacteria, we have started to investigate the marine alphaproteobacterium Hyphomonas neptunium (1), a representative of the stalked budding bacteria (2, 3). Similar to other members of this polyphyletic bacterial group, H. neptunium is characterized by a unique mode of reproduction that involves the formation of buds at the tip of a stalk emanating from the mother cell body (Fig. 1A). During the budding process, the nascent daughter cell is equipped with a single polar flagellum at the pole opposite the stalk. Cytokinesis then gives rise to a motile swarmer cell, which initially is unable to replicate, and an immotile stalked cell, which immediately enters a new round of budding and cell division (4). At a defined time in the cell cycle, the swarmer cell undergoes a differentiation process during which it sheds the flagellum and establishes a stalk at the opposite pole. Subsequently, a bud emerges from the tip of the stalk, setting the stage for the next division event.H. neptunium was isolated from the harbor of Barcelona (Spain). Based on morphological criteria, it was originally described as Hyphomicrobium neptunium (1). Later, DNA-DNA hybridization experiments, 5S rRNA sequence analyses, and metabolic profiling revealed a close phylogenetic relationship to members of the genus Hyphomonas (5, 6). Interestingly, 16S rRNA-based phylogenetic studies identify H. neptunium as a member of the Rhodobacterales (7). However, 23S rR...

The peptidoglycan cell wall of bacteria is a complex macromolecule composed of glycan strands that are cross-linked by short peptide bridges. Its biosynthesis involves a conserved group of enzymes, the bifunctional penicillin-binding proteins (bPBPs), which contain both a transglycosylase and a transpeptidase domain, thus being able to elongate the glycan strands and, at the same time, generate the peptide cross-links. The stalked model bacterium Caulobacter crescentus possesses five bPBP paralogs, named Pbp1A, PbpC, PbpX, PbpY, and PbpZ, whose function is still incompletely understood. In this study, we show that any of these proteins except for PbpZ is sufficient for growth and normal morphogenesis when expressed at native or elevated levels, whereas inactivation of all five paralogs is lethal. Growth analyses indicate a central role of PbpX in the resistance of C. crescentus against the noncanonical amino acid D-alanine. Moreover, we show that PbpX and PbpY localize to the cell division site. Their recruitment to the divisome is dependent on the essential cell division protein FtsN and likely involves interactions with FtsL and the putative peptidoglycan hydrolase DipM. The same interaction pattern is observed for Pbp1A and PbpC, although these proteins do not accumulate at midcell. Our findings demonstrate that the bPBPs of C. crescentus are, to a large extent, redundant and have retained the ability to interact with the peptidoglycan biosynthetic machineries responsible for cell elongation, cytokinesis, and stalk growth. Nevertheless, they may preferentially act in specific peptidoglycan biosynthetic complexes, thereby facilitating the independent regulation of distinct growth processes.