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ABSTRACT In most bacteria, cell division is centrally organized by the FtsZ protein, which assembles into dynamic filaments at the division site along the cell membrane that interact with other key cell division proteins. In gammaproteobacteria such as Escherichia coli, FtsZ filaments are anchored to the cell membrane by two essential proteins, FtsA and ZipA. Canonically, this interaction was believed to be mediated solely by the FtsZ C-terminal peptide (CTP) domain that interacts with these and several other regulatory proteins. However, we now provide evidence of a second interaction between FtsZ and ZipA. Using site-specific photoactivated cross-linking, we identified a noncanonical FtsZ-binding site on ZipA on the opposite side from the FtsZ CTP-binding pocket. Cross-linking at this site was unaffected by the truncation of the FtsZ linker and CTP domains, indicating that this noncanonical site must interact directly with the globular core domain of FtsZ. Mutations introduced into either the canonical or noncanonical binding sites on ZipA disrupted photo-cross-linking with FtsZ and normal ZipA function in cell division, suggesting that both binding modes are important for normal cell growth and division. One mutation at the noncanonical face was also found to suppress defects of several other canonical and noncanonical site mutations in ZipA, suggesting there is some interdependence between the two sites. Taken together, these results suggest that ZipA employs a two-pronged FtsZ-binding mechanism. IMPORTANCE The tubulin homolog FtsZ plays a central early role in organizing bacterial cell division proteins at the cytoplasmic membrane. However, FtsZ does not directly interact with the membrane itself, instead relying on proteins such as FtsA to tether it to the membrane. In gammaproteobacteria, ZipA serves as a second essential membrane anchor along with FtsA. Although FtsA has a unique role in activating synthesis of the cell division septum, and ZipA may in turn activate FtsA, it was thought that both proteins interacted only with the conserved C terminus of FtsZ and were essentially interchangeable in their ability to tether FtsZ to the membrane. Here we challenge this view, providing evidence that ZipA directly contacts both the C terminus and the core domain of FtsZ. Such a two-pronged interaction between ZipA and FtsZ suggests that ZipA and FtsA may serve distinct membrane-anchoring roles for FtsZ.

Growth and division are two of the most fundamental capabilities of a bacterial cell. While they are well described for model organisms growing in broth culture, very little is known about the cell division cycle of bacteria replicating in more complex environments. Using a D-alanine reporter strategy, we found that intracellular Listeria monocytogenes (Lm) spend a smaller proportion of their cell cycle dividing compared to Lm growing in broth culture. This alteration to the cell division cycle is independent of bacterial doubling time. Instead, polymerization of host-derived actin at the bacterial cell surface extends the non-dividing elongation period and compresses the division period. By decreasing the relative proportion of dividing Lm, actin polymerization biases the population toward cells with the highest propensity to form actin tails. Thus, there is a positive-feedback loop between the Lm cell division cycle and a physical interaction with the host cytoskeleton.

ABSTRACT The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales. To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales- and Rhodobacterales-specific l,d-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d-amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli. IMPORTANCE Many rod-shaped bacteria, including pathogens such as Brucella and Mycobacteriu, grow by adding new material to their cell poles, and yet the proteins and mechanisms contributing to this process are not yet well defined. The polarly growing plant pathogen Agrobacterium tumefaciens was used as a model bacterium to explore these polar growth mechanisms. The results obtained indicate that polar growth in this organism is facilitated by repurposed cell division components and an otherwise obscure class of alternative peptidoglycan transpeptidases (l,d-transpeptidases). This growth results in dynamically changing cell widths as the poles expand to maturity and contrasts with the tightly regulated cell widths characteristic of canonical rod-shaped growth. Furthermore, the abundance and/or activity of l,d-transpeptidases appears to associate with polar growth strategies, suggesting that these enzymes may serve as attractive targets for specifically inhibiting growth of Rhizobiales, Actinomycetales, and other polarly growing bacterial pathogens.

ABSTRACT Type IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain of Agrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in the A. tumefaciens octopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression following vir induction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles. vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiple vir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates. IMPORTANCE Transfer of DNA and/or proteins to host cells through multiprotein type IV secretion system (T4SS) complexes that span the bacterial cell envelope is critical to bacterial pathogenesis. Early reports suggested that T4SS components localized at the cell poles. Now, higher-resolution deconvolution fluorescence microscopy reveals that all structural components of the Agrobacterium tumefaciens vir-T4SS, as well as its transported protein substrates, localize to multiple foci around the cell perimeter. These results lead to a new model of A. tumefaciens attachment to a plant cell, where A. tumefaciens takes advantage of the multiple vir-T4SS along its length to make intimate lateral contact with plant cells and thereby effectively transfer DNA and/or proteins through the vir-T4SS. The T4SS of A. tumefaciens is among the best-studied T4SS, and the majority of its components are highly conserved in different pathogenic bacterial species. Thus, the results presented can be applied to a broad range of pathogens that utilize T4SS.

Treponema pallidum ssp. pallidum, the causative agent of syphilis, can now be cultured continuously in vitro utilizing a tissue culture system, and the multiplication rates are similar to those obtained in experimental infection of rabbits. In this study, the RNA transcript profiles of the T. pallidum Nichols during in vitro culture and rabbit infection were compared to examine whether gene expression patterns differed in these two environments. To this end, RNA preparations were converted to cDNA and subjected to RNA-seq using high throughput Illumina sequencing; reverse transcriptase quantitative PCR was also performed on selected genes for validation of results. The transcript profiles in the in vivo and in vitro environments were remarkably similar, exhibiting a high degree of concordance overall. However, transcript levels of 94 genes (9%) out of the 1,063 predicted genes in the T. pallidum genome were significantly different during rabbit infection versus in vitro culture, varying by up to 8-fold in the two environments. Genes that exhibited significantly higher transcript levels during rabbit infection included those encoding multiple ribosomal proteins, several prominent membrane proteins, glycolysis-associated enzymes, replication initiator DnaA, rubredoxin, thioredoxin, two putative regulatory proteins, and proteins associated with solute transport. In vitro cultured T. pallidum had higher transcript levels of DNA repair proteins, cofactor synthesis enzymes, and several hypothetical proteins. The overall concordance of the transcript profiles may indicate that these environments are highly similar in terms of their effects on T. pallidum physiology and growth, and may also reflect a relatively low level of transcriptional regulation in this reduced genome organism.

Almost 60 years ago, Severo Ochoa was awarded the Nobel Prize in Physiology or Medicine for his discovery of the enzymatic synthesis of RNA by polynucleotide phosphorylase (PNPase). Although this discovery provided an important tool for deciphering the genetic code, subsequent work revealed that the predominant function of PNPase in bacteria and eukaryotes is catalyzing the reverse reaction, i.e., the release of ribonucleotides from RNA. PNPase has a crucial role in RNA metabolism in bacteria and eukaryotes mainly through its roles in processing and degrading RNAs, but additional functions in RNA metabolism have recently been reported for this enzyme. Here, we discuss these established and noncanonical functions for PNPase and the possibility that the major impact of PNPase on cell physiology is through its unorthodox roles.

The Gram negative plant pathogen Agrobacterium tumefaciens is uniquely capable of genetically transforming eukaryotic host cells during the infection process. DNA and protein substrates are transferred into plant cells via a type IV secretion system (T4SS), which forms large cell-envelope spanning complexes at multiple sites around the bacterial circumference. To gain a detailed understanding of T4SS positioning, the spatial distribution of fluorescently labeled T4SS components was quantitatively assessed to distinguish between random and structured localization processes. Through deconvolution microscopy followed by Fourier analysis and modeling, T4SS foci were found to localize in a non-random periodic pattern. These results indicate that T4SS complexes are dependent on an underlying scaffold or assembly process to obtain an organized distribution suitable for effective delivery of substrates into host cells.