In the environment bacteria share their habitat with a great diversity of organisms, from microbes to humans, animals and plants. In these complex communities, the production of extracellular effectors is a common strategy to control the biodiversity by interfering with the growth and/or viability of nearby microbes. One of such effectors relies on the production and release of extracellular D-amino acids which regulate diverse cellular processes such as cell wall biogenesis, biofilm integrity, and spore germination. Non-canonical D-amino acids are mainly produced by broad spectrum racemases (Bsr). Bsr’s promiscuity allows it to generate high concentrations of D-amino acids in environments with variable compositions of L-amino acids. However, it was not clear until recent whether these molecules exhibit divergent functions. Here we review the distinctive biological roles of D-amino acids, their mechanisms of action and their modulatory properties of the biodiversity of complex eco-systems.

Bacteria face tough competition in polymicrobial communities. To persist in a specific niche, many species produce toxic extracellular effectors to interfere with the growth of nearby microbes. These effectors include the recently reported non-canonical D-amino acids (NCDAAs). In Vibrio cholerae, the causative agent of cholera, NCDAAs control cell wall integrity in stationary phase. Here, an analysis of the composition of the extracellular medium of V. cholerae revealed the unprecedented presence of D-Arg. Compared with other D-amino acids, D-Arg displayed higher potency and broader toxicity in terms of the number of bacterial species affected. Tolerance to D-Arg was associated with mutations in the phosphate transport and chaperone systems, whereas D-Met lethality was suppressed by mutations in cell wall determinants. These observations suggest that NCDAAs target different cellular processes. Finally, even though virtually all Vibrio species are tolerant to D-Arg, only a few can produce this D-amino acid. Indeed, we demonstrate that D-Arg may function as part of a cooperative strategy in vibrio communities to protect non-producing members from competing bacteria. Because NCDAA production is widespread in bacteria, we anticipate that D-Arg is a relevant modulator of microbial subpopulations in diverse ecosystems.

The peptidoglycan (PG) cell wall is an essential polymer for the shape and viability of bacteria. Its protective role is in great part provided by its mesh-like character. Therefore, PG-cross-linking enzymes like the penicillin-binding proteins (PBPs) are among the best targets for antibiotics. However, while PBPs have been in the spotlight for more than 50 years, another class of PG-cross-linking enzymes called LDtranspeptidases (LDTs) seemed to contribute less to PG synthesis and, thus, has kept an aura of mystery. In the last years, a number of studies have associated LDTs with cell wall adaptation to stress including blactam antibiotics, outer membrane stability, and toxin delivery, which has shed light onto the biological meaning of these proteins. Furthermore, as some species display a great abundance of LD-cross-links in their cell wall, it has been hypothesized that LDTs could also be the main synthetic PGtranspeptidases in some bacteria. In this review, we introduce these enzymes and their role in PG biosynthesis and we highlight the most recent advances in understanding their biological role in diverse species.

Agrobacterium tumefaciens is a rod-shaped bacterium that grows by polar insertion of new peptidoglycan during cell elongation. As the cell cycle progresses, peptidoglycan synthesis at the pole ceases prior to insertion of new peptidoglycan at midcell to enable cell division. The A. tumefaciens homolog of the Caulobacter crescentus polar organelle development protein PopZ has been identified as a growth pole marker and a candidate polar growth-promoting factor. Here, we characterize the function of PopZ in cell growth and division of A. tumefaciens. Consistent with previous observations, we observe that PopZ localizes specifically to the growth pole in wild-type cells. Despite the striking localization pattern of PopZ, we find the absence of the protein does not impair polar elongation or cause major changes in the peptidoglycan composition. Instead, we observe an atypical cell length distribution, including minicells, elongated cells, and cells with ectopic poles. Most minicells lack DNA, suggesting a defect in chromosome segregation. Furthermore, the canonical cell division proteins FtsZ and FtsA are misplaced, leading to asymmetric sites of cell constriction. Together, these data suggest that PopZ plays an important role in the regulation of chromosome segregation and cell division.IMPORTANCE A. tumefaciens is a bacterial plant pathogen and a natural genetic engineer. However, very little is known about the spatial and temporal regulation of cell wall biogenesis that leads to polar growth in this bacterium. Understanding the molecular basis of A. tumefaciens growth may allow for the development of innovations to prevent disease or to promote growth during biotechnology applications. Finally, since many closely related plant and animal pathogens exhibit polar growth, discoveries in A. tumefaciens may be broadly applicable for devising antimicrobial strategies.KEYWORDS Agrobacterium, PopZ, cell polarity, chromosome segregation, cell division, growth polarity A grobacterium tumefaciens, the causative agent of crown gall disease in flowering plants, has been studied extensively with regard to pathogenesis and its ability to transfer engineered DNA to plant cells using the type IV secretion system encoded by multiple vir genes. More recent observations of A. tumefaciens growth have revealed that this bacterium exhibits polar growth during elongation (1, 2). In A. tumefaciens, as well as in other Rhizobiales, peptidoglycan is inserted at the new poles created by cell division until the cell doubles in length. The peptidoglycan synthesis is then directed to midcell to enable septum formation and cell division. The growth patterning of A.

Summary The mechanisms that restrict peptidoglycan biosynthesis to the pole during elongation and re‐direct peptidoglycan biosynthesis to mid‐cell during cell division in polar‐growing Alphaproteobacteria are largely unknown. Here, we explore the role of early division proteins of Agrobacterium tumefaciens including three FtsZ homologs, FtsA and FtsW in the transition from polar growth to mid‐cell growth and ultimately cell division. Although two of the three FtsZ homologs localize to mid‐cell, exhibit GTPase activity and form co‐polymers, only one, FtsZAT, is required for cell division. We find that FtsZAT is required not only for constriction and cell separation, but also for initiation of peptidoglycan synthesis at mid‐cell and cessation of polar peptidoglycan biosynthesis. Depletion of FtsZAT in A. tumefaciens causes a striking phenotype: cells are extensively branched and accumulate growth active poles through tip splitting events. When cell division is blocked at a later stage by depletion of FtsA or FtsW, polar growth is terminated and ectopic growth poles emerge from mid‐cell. Overall, this work suggests that A. tumefaciens FtsZ makes distinct contributions to the regulation of polar growth and cell division.

While the structure and function of the bacterial cell wall are well conserved, the mechanisms responsible for cell wall biosynthesis during elongation are variable. It is increasingly clear that rod-shaped bacteria use a diverse array of growth strategies with distinct spatial zones of cell wall biosynthesis, including lateral elongation, unipolar growth, bipolar elongation, and medial elongation.

11The mechanisms that restrict peptidoglycan biosynthesis to the pole during elongation and re-12 direct peptidoglycan biosynthesis to mid-cell during cell division in polar-growing 13 Alphaproteobacteria are largely unknown. Here, we demonstrate that although two of the three 14 FtsZ homologs localize to mid-cell, exhibit GTPase activity and form co-polymers, only one, 15 FtsZAT, is required for cell division. We find that FtsZAT is required not only for constriction and 16 cell separation, but also for the termination of polar growth and regulation of peptidoglycan 17 synthesis at mid-cell. Depletion of FtsZ in A. tumefaciens causes a striking phenotype: cells are 18 extensively branched and accumulate growth active poles through tip splitting events. When cell 19 division is blocked at a later stage, polar growth is terminated and ectopic growth poles emerge 20 from mid-cell. Overall, this work suggests that A. tumefaciens FtsZ makes distinct contributions 21 to the regulation of polar growth and cell division. 23To initiate bacterial cell division, the tubulin-like GTPase, FtsZ, polymerizes and forms a 33 discontinuous ring-like structure at the future site of cell division [4][5][6][7][8][9][10]. The presence of FtsZ at 34 mid-cell leads to the recruitment of many proteins that function in cell division, collectively 35 called the divisome [11][12][13][14]. The divisome includes cell wall biosynthesis proteins, such as the 36 penicillin-binding protein, PBP3, and FtsW, which contribute to PG biosynthesis and remodeling 37 necessary to form new poles in daughter cells [11]. Once the divisome is fully assembled, FtsZ 38 filaments treadmill along the circumference of the mid-cell, driving the Z-ring constriction [9, 39 10]. The movement of FtsZ filaments is correlated with the movement of enzymes that function 40 in septal PG biogenesis. These finding are consistent with the notion that FtsZ not only recruits 41 enzymes that function in PG biogenesis to mid-cell but also regulates their activities to promote 42 proper cell wall biogenesis [15-17].43 44In most rod-shaped model organisms used to study cell division, a block in cell division leads to 45 the production of long, smooth filamentous cells. This phenotype suggests that assembly or 46 activation of some divisome components is necessary not only to enable the cells to divide but 47 also to stop cellular elongation. Indeed, in Escherichia coli, FtsZ (along with the Z-ring 48 stabilizing proteins FtsA, ZipA, and ZapA) has been proposed to have an early function in the 49 switch from lateral PG biogenesis to mid-cell PG biosynthesis [18]. Following maturation of the 50 divisome by recruitment of additional PG remodeling enzymes and cell division proteins, PG 51 biosynthesis is coordinated with membrane invagination, enabling cells to constrict and separate 52 [19].53 54 4Conversely to e.g. E. coli, polar growing rods in the alphaproteobacterial clade Rhizobiales 55 exhibit branched morphologies when cell division is blocked [20][21][22][23][24][...

Members of the Rhizobiales are polarly-growing bacteria that lack homologs of the canonical rod complex. To investigate the mechanisms underlying polar cell wall synthesis, we systematically probed the function of cell wall synthesis enzymes in the plant-pathogen Agrobacterium tumefaciens. The development of fluorescent D-amino acid dipeptide (FDAAD) probes, which are incorporated into peptidoglycan by penicillin-binding proteins in A. tumefaciens, enabled us to monitor changes in growth patterns in the mutants. Use of these fluorescent cell wall probes and peptidoglycan compositional analysis convincingly demonstrate that a single class A penicillin-binding protein is essential for polar peptidoglycan synthesis. Furthermore, we find evidence of an alternative mode of cell wall synthesis that likely requires LD-transpeptidase activity. Genetic analysis and cell wall targeting antibiotics reveal that the mechanism of unipolar growth is conserved in Sinorhizobium and Brucella. This work provides insights into unipolar peptidoglycan biosynthesis employed by the Rhizobiales during cell elongation.