The divergent early divisome: is there a functional core?

The bacterial divisome is a complex nanomachine that drives cell division and separation. The essentiality of these processes leads to the assumption that proteins with core roles will be strictly conserved across all bacterial genomes. However, recent studies in diverse proteobacteria have revealed considerable variation in the early divisome compared with Escherichia coli. While some proteins are highly conserved, their specific functions and interacting partners vary. Meanwhile, different subphyla use clade-specific proteins with analogous functions. Thus, instead of focusing on gene conservation, we must also explore how key functions are maintained during early division by diverging protein networks. An enhanced awareness of these complex genetic networks will clarify the physical and evolutionary constraints of bacterial division.

Diversification of LytM Protein Functions in Polar Elongation and Cell Division of Agrobacterium tumefaciens.

LytM-domain containing proteins are LAS peptidases (lysostaphin-type enzymes, D-Ala-D-Ala metallopeptidases, and sonic hedgehog) and are known to play diverse roles throughout the bacterial cell cycle through direct or indirect hydrolysis of the bacterial cell wall. A subset of the LytM factors are catalytically inactive but regulate the activity of other cell wall hydrolases and are classically described as cell separation factors NlpD and EnvC. Here, we explore the function of four LytM factors in the alphaproteobacterial plant pathogen Agrobacterium tumefaciens. An LmdC ortholog (Atu1832) and a MepM ortholog (Atu4178) are predicted to be catalytically active. While Atu1832 does not have an obvious function in cell growth or division, Atu4178 is essential for polar growth and likely functions as a space-making endopeptidase that cleaves amide bonds in the peptidoglycan cell wall during elongation. The remaining LytM factors are degenerate EnvC and NlpD orthologs. Absence of these proteins results in striking phenotypes indicative of misregulation of cell division and growth pole establishment. The deletion of an amidase, AmiC, closely phenocopies the deletion of envC suggesting that EnvC might regulate AmiC activity. The NlpD ortholog DipM is unprecedently essential for viability and depletion results in the misregulation of early stages of cell division, contrasting with the canonical view of DipM as a cell separation factor. Finally, we make the surprising observation that absence of AmiC relieves the toxicity induced by dipM overexpression. Together, these results suggest EnvC and DipM may function as regulatory hubs with multiple partners to promote proper cell division and establishment of polarity.

An Essential Regulator of Bacterial Division Links FtsZ to Cell Wall Synthase Activation.

Bacterial growth and division require insertion of new peptidoglycan (PG) into the existing cell wall by PG synthase enzymes. Emerging evidence suggests that many PG synthases require activation to function; however, it is unclear how activation of division-specific PG synthases occurs. The FtsZ cytoskeleton has been implicated as a regulator of PG synthesis during division, but the mechanisms through which it acts are unknown. Here, we show that FzlA, an FtsZ-binding protein and essential regulator of constriction in Caulobacter crescentus, helps link FtsZ to PG synthesis to promote division. We find that hyperactive mutants of the PG synthases FtsW and FtsI specifically render fzlA, but not other division genes, non-essential. However, FzlA is still required to maintain proper constriction rate and efficiency in a hyperactive PG synthase background. Intriguingly, loss of fzlA in the presence of hyperactivated FtsWI causes cells to rotate about the division plane during constriction and sensitizes cells to cell-wall-specific antibiotics. We demonstrate that FzlA-dependent signaling to division-specific PG synthesis is conserved in another α-proteobacterium, Agrobacterium tumefaciens. These data establish that FzlA helps link FtsZ to cell wall remodeling and is required for signaling to both activate and spatially orient PG synthesis during division. Overall, our findings support the paradigm that activation of SEDS-PBP PG synthases is a broadly conserved requirement for bacterial morphogenesis.

Para-Substituted Functionalised Ferrocene Esters with Novel Antibacterial Properties.

INTRODUCTION: Bacterial antibiotic resistance is on rise despite advances in the development of new antibiotics. In an attempt to circumvent resistance, scientists are shifting focus from modifying existent antibiotics to identifying new antibiotic compounds. AIM: To assess the potential antibiotic effects of functionalised ferrocenecarboxylates para-substituted on the phenoxy pendant group to form: 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-iodophenyl and 4-(H-pyrrol-1-yl)phenyl. MATERIALS AND METHODS: For this, we employed the Kirby-Bauer disc diffusion method using a collection of nine bacterial species: Staphylococcus aureus, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Serratia marcescens, Klebsiella pneumoniae, Bacillus subtilis, Proteus vulgaris and Enterobacter aerogenes. RESULTS: The results show that all four-halogen substituted ferrocenecarboxylates 4-fluorophenyl (23.33 µM, 11.66 µM, 5.83 µM), 4-chlorophenyl (10.16 µM, 5.08 µM, 2.54 µM), 4-bromophenyl (9.0 µM, 4.5 µM, 2.25 µM), and 4-iodophenyl (17.12 µM, 8.56 µM, 4.28 µM) exhibited an antibacterial effect by reducing proliferation of Bacillus subtilis. Meanwhile, only 4-bromophenyl (9.0 µM) and 4-chlorophenyl (10.16 µM) ferrocenecarboxylates were able to decrease the growth of Micrococcus luteus. CONCLUSION: Hence, functionalised ferrocenecarboxylates para-substituted with small and simple groups represent a novel class of bio-organometallic compounds with the potential to be used as antibacterial agents.

Domains within RbpA Serve Specific Functional Roles That Regulate the Expression of Distinct Mycobacterial Gene Subsets.

The RNA polymerase (RNAP) binding protein A (RbpA) contributes to the formation of stable RNAP-promoter open complexes (RPo) and is essential for viability in mycobacteria. Four domains have been identified in the RbpA protein, i.e., an N-terminal tail (NTT) that interacts with RNAP ß' and σ subunits, a core domain (CD) that contacts the RNAP ß' subunit, a basic linker (BL) that binds DNA, and a σ-interaction domain (SID) that binds group I and group II σ factors. Limited in vivo studies have been performed in mycobacteria, however, and how individual structural domains of RbpA contribute to RbpA function and mycobacterial gene expression remains mostly unknown. We investigated the roles of the RbpA structural domains in mycobacteria using a panel of rbpA mutants that target individual RbpA domains. The function of each RbpA domain was required for Mycobacterium tuberculosis viability and optimal growth in Mycobacterium smegmatis We determined that the RbpA SID is both necessary and sufficient for RbpA interaction with the RNAP, indicating that the primary functions of the NTT and CD are not solely association with the RNAP. We show that the RbpA BL and SID are required for RPo stabilization in vitro, while the NTT and CD antagonize this activity. Finally, RNA-sequencing analyses suggest that the NTT and CD broadly activate gene expression, whereas the BL and SID activate or repress gene expression in a gene-dependent manner for a subset of mycobacterial genes. Our findings highlight specific outcomes for the activities of the individual functional domains in RbpA.IMPORTANCEMycobacterium tuberculosis is the causative agent of tuberculosis and continues to be the most lethal infectious disease worldwide. Improved molecular understanding of the essential proteins involved in M. tuberculosis transcription, such as RbpA, could provide targets for much needed future therapeutic agents aimed at combatting this pathogen. In this study, we expand our understanding of RbpA by identifying the RbpA structural domains responsible for the interaction of RbpA with the RNAP and the effects of RbpA on transcription initiation and gene expression. These experiments expand our knowledge of RbpA while also broadening our understanding of bacterial transcription in general.