Tracking a bug’s life: We describe the first direct and universal approach for labeling peptidoglycan (PG) of diverse bacteria by exploiting the surprising tolerance of cells for incorporating unnatural D-amino acids of various sizes and functionalities. These non-toxic D-amino acids preferably label the sites of active PG synthesis, enabling fine spatiotemporal tracking of cell wall dynamics in phylogenetically and morphologically diverse bacteria.

Elongation of many rod-shaped bacteria occurs by peptidoglycan synthesis at discrete foci along the sidewall of the cells. However, within the Rhizobiales, there are many budding bacteria, in which new cell growth is constrained to a specific region. The phylogeny of the Rhizobiales indicates that this mode of zonal growth may be ancestral. We demonstrate that the rod-shaped bacterium Agrobacterium tumefaciens grows unidirectionally from the new pole generated after cell division and has an atypical peptidoglycan composition. Polar growth occurs under all conditions tested, including when cells are attached to a plant root and under conditions that induce virulence. Finally, we show that polar growth also occurs in the closely related bacteria Sinorhizobium meliloti , Brucella abortus , and Ochrobactrum anthropi . We find that unipolar growth is an ancestral and conserved trait among the Rhizobiales, which includes important mutualists and pathogens of plants and animals.

Summary The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide-mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili-dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just-in-time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non-productive self-adherence, and preventing curing of the adhesive.

SummaryCaulobacter crescentus attachment is mediated by the holdfast, a complex of polysaccharide anchored to the cell by HfaA, HfaB and HfaD. We show that all three proteins are surface exposed outer membrane (OM) proteins. HfaA is similar to fimbrial proteins and assembles into a high molecular weight (HMW) form requiring HfaD, but not holdfast polysaccharide. The HfaD HMW form is dependent on HfaA but not on holdfast polysaccharide. We show that HfaA and HfaD form homomultimers and that they require HfaB for stability and OM translocation. All three proteins localize to the late pre-divisional flagellar pole, remain at this pole in swarmer cells, and localize at the stalk tip after the stalk is synthesized at the same pole. Hfa protein localization requires the holdfast polysaccharide secretion proteins and the polar localization factor PodJ. An hfaB mutant is much more severely deficient in adherence and holdfast attachment than hfaA and hfaD mutants. An hfaA, hfaD double mutant phenocopies either single mutant, suggesting that HfaB is involved in holdfast attachment beyond secretion of HfaA and HfaD. We hypothesize that HfaB secretes HfaA and HfaD across the outer membrane, and the three proteins form a complex anchoring the holdfast to the stalk.

What mechanisms underlie the transitions responsible for the diverse shapes observed in the living world? While bacteria display a myriad of morphologies1, the mechanisms responsible for the evolution of bacterial cell shape are not understood. We investigated morphological diversity in a group of bacteria that synthesize an appendage-like extension of the cell envelope called the stalk2,3. The location and number of stalks varies among species, as exemplified by three distinct sub-cellular positions of stalks within a rod-shaped cell body: polar in the Caulobacter genus, and sub-polar or bi-lateral in the Asticcacaulis genus4. Here we show that a developmental regulator of Caulobacter crescentus, SpmX5, was co-opted in the Asticcacaulis genus to specify stalk synthesis at either the sub-polar or bi-lateral positions. We show that stepwise evolution of a specific region of SpmX led to the gain of a new function and localization of this protein, which drove the sequential transition in stalk positioning. Our results indicate that evolution of protein function, co-option, and modularity are key elements in the evolution of bacterial morphology. Therefore, similar evolutionary principles of morphological transitions apply to both single-celled prokaryotes and multicellular eukaryotes.