mSphere of Influence: Celebrating exceptions to the rule of lipid A essentiality.

Kate Hummels works in the field of bacterial cell envelope biosynthesis and studies the regulation of the metabolic pathways needed to build the Gram-negative cell envelope. In this mSphere of Influence article, she reflects on how the papers "A penicillin-binding protein inhibits selection of colistin-resistant, lipopoligosaccharide-deficient Acinetobacter baumannii" by Boll et al. and "Caulobacter lipid A is conditionally dispensable in the absence of fur and in the presence of anionic sphingolipids" by Zik et al. made an impact on her by studying organisms that deviate from accepted norms to highlight the plethora of unanswered questions in cell envelope biology.

Visualizing dynamic competence pili and DNA capture throughout the long axis of Bacillus subtilis.

The first step in the process of bacterial natural transformation is DNA capture. Although long hypothesized based on genetics and functional experiments, the pilus structure responsible for initial DNA binding had not yet been visualized for Bacillus subtilis. Here, we visualize functional competence pili in Bacillus subtilis using fluorophore-conjugated maleimide labeling in conjunction with epifluorescence microscopy. In strains that produce pilin monomers within tenfold of wild-type levels, the median length of detectable pili is 300 nm. These pili are retractile and associate with DNA. The analysis of pilus distribution at the cell surface reveals that they are predominantly located along the long axis of the cell. The distribution is consistent with localization of proteins associated with subsequent transformation steps, DNA binding, and DNA translocation in the cytosol. These data suggest a distributed model for B. subtilis transformation machinery, in which initial steps of DNA capture occur throughout the long axis of the cell and subsequent steps may also occur away from the cell poles. IMPORTANCE This work provides novel visual evidence for DNA translocation across the cell wall during Bacillus subtilis natural competence, an essential step in the natural transformation process. Our data demonstrate the existence of natural competence-associated retractile pili that can bind exogenous DNA. Furthermore, we show that pilus biogenesis occurs throughout the cell long axis. These data strongly support DNA translocation occurring all along the lateral cell wall during natural competence, wherein pili are produced, bind to free DNA in the extracellular space, and finally retract to pull the bound DNA through the gap in the cell wall created during pilus biogenesis.

Visualizing dynamic competence pili and DNA capture throughout the long axis of Bacillus subtilis.

The first step in the process of bacterial natural transformation is DNA capture. Although long-hypothesized based on genetics and functional experiments, the pilus structure responsible for initial DNA-binding had not yet been visualized for Bacillus subtilis. Here, we visualize functional competence pili in Bacillus subtilis using fluorophore-conjugated maleimide labeling in conjunction with epifluorescence microscopy. In strains that produce pilin monomers within ten-fold of wild type levels, the median length of detectable pili is 300nm. These pili are retractile and associate with DNA. Analysis of pilus distribution at the cell surface reveals that they are predominantly located along the long axis of the cell. The distribution is consistent with localization of proteins associated with subsequent transformation steps, DNA-binding and DNA translocation in the cytosol. These data suggest a distributed model for B. subtilis transformation machinery, in which initial steps of DNA capture occur throughout the long axis of the cell and subsequent steps may also occur away from the cell poles.

Coordination of bacterial cell wall and outer membrane biosynthesis.

Gram-negative bacteria surround their cytoplasmic membrane with a peptidoglycan (PG) cell wall and an outer membrane (OM) with an outer leaflet composed of lipopolysaccharide (LPS)1. This complex envelope presents a formidable barrier to drug entry and is a major determinant of the intrinsic antibiotic resistance of these organisms2. The biogenesis pathways that build the surface are also targets of many of our most effective antibacterial therapies3. Understanding the molecular mechanisms underlying the assembly of the Gram-negative envelope therefore promises to aid the development of new treatments effective against the growing problem of drug-resistant infections. Although the individual pathways for PG and OM synthesis and assembly are well characterized, almost nothing is known about how the biogenesis of these essential surface layers is coordinated. Here we report the discovery of a regulatory interaction between the committed enzymes for the PG and LPS synthesis pathways in the Gram-negative pathogen Pseudomonas aeruginosa. We show that the PG synthesis enzyme MurA interacts directly and specifically with the LPS synthesis enzyme LpxC. Moreover, MurA was shown to stimulate LpxC activity in cells and in a purified system. Our results support a model in which the assembly of the PG and OM layers in many proteobacterial species is coordinated by linking the activities of the committed enzymes in their respective synthesis pathways.

Translation elongation factor P (EF-P).

Translation elongation factor P (EF-P) is conserved in all three domains of life (called eIF5A and aIF5A in eukaryotes and archaea, respectively) and functions to alleviate ribosome pausing during the translation of specific sequences, including consecutive proline residues. EF-P was identified in 1975 as a factor that stimulated the peptidyltransferase reaction in vitro but its involvement in the translation of tandem proline residues was not uncovered until 2013. Throughout the four decades of EF-P research, perceptions of EF-P function have changed dramatically. In particular, while EF-P was thought to potentiate the formation of the first peptide bond in a protein, it is now broadly accepted to act throughout translation elongation. Further, EF-P was initially reported to be essential, but recent work has shown that the requirement of EF-P for growth is conditional. Finally, it is thought that post-translational modification of EF-P is strictly required for its function but recent studies suggest that EF-P modification may play a more nuanced role in EF-P activity. Here, we review the history of EF-P research, with an emphasis on its initial isolation and characterization as well as the discoveries that altered our perceptions of its function.

Suppressor mutations in ribosomal proteins and FliY restore Bacillus subtilis swarming motility in the absence of EF-P.

Translation elongation factor P (EF-P) alleviates ribosome pausing at a subset of motifs encoding consecutive proline residues, and is required for growth in many organisms. Here we show that Bacillus subtilis EF-P also alleviates ribosome pausing at sequences encoding tandem prolines and ribosomes paused within several essential genes without a corresponding growth defect in an efp mutant. The B. subtilis efp mutant is instead impaired for flagellar biosynthesis which results in the abrogation of a form of motility called swarming. We isolate swarming suppressors of efp and identify mutations in 8 genes that suppressed the efp mutant swarming defect, many of which encode conserved ribosomal proteins or ribosome-associated factors. One mutation abolished a translational pause site within the flagellar C-ring component FliY to increase flagellar number and restore swarming motility in the absence of EF-P. Our data support a model wherein EF-P-alleviation of ribosome pausing may be particularly important for macromolecular assemblies like the flagellum that require precise protein stoichiometries.

A bifunctional ATPase drives tad pilus extension and retraction.

A widespread class of prokaryotic motors powered by secretion motor adenosine triphosphatases (ATPases) drives the dynamic extension and retraction of extracellular fibers, such as type IV pili (T4P). Among these, the tight adherence (tad) pili are critical for surface sensing and biofilm formation. As for most other motors belonging to this class, how tad pili retract despite lacking a dedicated retraction motor ATPase has remained a mystery. Here, we find that a bifunctional pilus motor ATPase, CpaF, drives both activities through adenosine 5'-triphosphate (ATP) hydrolysis. We show that mutations within CpaF result in a correlated reduction in the rates of extension and retraction that directly scales with decreased ATP hydrolysis and retraction force. Thus, a single motor ATPase drives the bidirectional processes of pilus fiber extension and retraction.

EF-P Posttranslational Modification Has Variable Impact on Polyproline Translation in Bacillus subtilis.

Elongation factor P (EF-P) is a ubiquitous translation factor that facilitates translation of polyproline motifs. In order to perform this function, EF-P generally requires posttranslational modification (PTM) on a conserved residue. Although the position of the modification is highly conserved, the structure can vary widely between organisms. In Bacillus subtilis, EF-P is modified at Lys32 with a 5-aminopentanol moiety. Here, we use a forward genetic screen to identify genes involved in 5-aminopentanolylation. Tandem mass spectrometry analysis of the PTM mutant strains indicated that ynbB, gsaB, and ymfI are required for modification and that yaaO, yfkA, and ywlG influence the level of modification. Structural analyses also showed that EF-P can retain unique intermediate modifications, suggesting that 5-aminopentanol is likely directly assembled on EF-P through a novel modification pathway. Phenotypic characterization of these PTM mutants showed that each mutant does not strictly phenocopy the efp mutant, as has previously been observed in other organisms. Rather, each mutant displays phenotypic characteristics consistent with those of either the efp mutant or wild-type B. subtilis depending on the growth condition. In vivo polyproline reporter data indicate that the observed phenotypic differences result from variation in both the severity of polyproline translation defects and altered EF-P context dependence in each mutant. Together, these findings establish a new EF-P PTM pathway and also highlight a unique relationship between EF-P modification and polyproline context dependence.IMPORTANCE Despite the high level of conservation of EF-P, the posttranslational modification pathway that activates EF-P is highly divergent between species. Here, we have identified and characterized in B. subtilis a novel posttranslational modification pathway. This pathway not only broadens the scope of potential EF-P modification strategies, but it also indicates that EF-P modifications can be assembled directly on EF-P. Furthermore, characterization of these PTM mutants has established that an altered modification state can impact both the severity of polyproline translational defects and context dependence.

Carbonyl reduction by YmfI in Bacillus subtilis prevents accumulation of an inhibitory EF-P modification state.

Translation elongation factor P (EF-P) in Bacillus subtilis is required for a form of surface migration called swarming motility. Furthermore, B. subtilis EF-P is post-translationally modified with a 5-aminopentanol group but the pathway necessary for the synthesis and ligation of the modification is unknown. Here we determine that the protein YmfI catalyzes the reduction of EF-P-5 aminopentanone to EF-P-5 aminopentanol. In the absence of YmfI, accumulation of 5-aminopentanonated EF-P is inhibitory to swarming motility. Suppressor mutations that enhanced swarming in the absence of YmfI were found at two positions on EF-P, including one that changed the conserved modification site (Lys 32) and abolished post-translational modification. Thus, while modification of EF-P is thought to be essential for EF-P activity, here we show that in some cases it can be dispensable. YmfI is the first protein identified in the pathway leading to EF-P modification in B. subtilis, and B. subtilis encodes the first EF-P ortholog that retains function in the absence of modification.

Translation Control of Swarming Proficiency in Bacillus subtilis by 5-Amino-pentanolylated Elongation Factor P.

Elongation factor P (EF-P) accelerates diprolyl synthesis and requires a posttranslational modification to maintain proteostasis. Two phylogenetically distinct EF-P modification pathways have been described and are encoded in the majority of Gram-negative bacteria, but neither is present in Gram-positive bacteria. Prior work suggested that the EF-P-encoding gene (efp) primarily supports Bacillus subtilis swarming differentiation, whereas EF-P in Gram-negative bacteria has a more global housekeeping role, prompting our investigation to determine whether EF-P is modified and how it impacts gene expression in motile cells. We identified a 5-aminopentanol moiety attached to Lys(32) of B. subtilis EF-P that is required for swarming motility. A fluorescent in vivo B. subtilis reporter system identified peptide motifs whose efficient synthesis was most dependent on 5-aminopentanol EF-P. Examination of the B. subtilis genome sequence showed that these EF-P-dependent peptide motifs were represented in flagellar genes. Taken together, these data show that, in B. subtilis, a previously uncharacterized posttranslational modification of EF-P can modulate the synthesis of specific diprolyl motifs present in proteins required for swarming motility.

Specificity residues determine binding affinity for two-component signal transduction systems.

UNLABELLED: Two-component systems (TCS) comprise histidine kinases and their cognate response regulators and allow bacteria to sense and respond to a wide variety of signals. Histidine kinases (HKs) phosphorylate and dephosphorylate their cognate response regulators (RRs) in response to stimuli. In general, these reactions appear to be highly specific and require an appropriate association between the HK and RR proteins. The Myxococcus xanthus genome encodes one of the largest repertoires of signaling proteins in bacteria (685 open reading frames [ORFs]), including at least 127 HKs and at least 143 RRs. Of these, 27 are bona fide NtrC-family response regulators, 21 of which are encoded adjacent to their predicted cognate kinases. Using system-wide profiling methods, we determined that the HK-NtrC RR pairs display a kinetic preference during both phosphotransfer and phosphatase functions, thereby defining cognate signaling systems in M. xanthus. Isothermal titration calorimetry measurements indicated that cognate HK-RR pairs interact with dissociation constants (Kd) of approximately 1 µM, while noncognate pairs had no measurable binding. Lastly, a chimera generated between the histidine kinase, CrdS, and HK1190 revealed that residues conferring phosphotransfer and phosphatase specificity dictate binding affinity, thereby establishing discrete protein-protein interactions which prevent cross talk. The data indicate that binding affinity is a critical parameter governing system-wide signaling fidelity for bacterial signal transduction proteins. IMPORTANCE: Using in vitro phosphotransfer and phosphatase profiling assays and isothermal titration calorimetry, we have taken a system-wide approach to demonstrate specificity for a family of two-component signaling proteins in Myxococcus xanthus. Our results demonstrate that previously identified specificity residues dictate binding affinity and that phosphatase specificity follows phosphotransfer specificity for cognate HK-RR pairs. The data indicate that preferential binding affinity is the basis for signaling fidelity in bacterial two-component systems.

Draft Genome Sequence of Myxococcus xanthus Wild-Type Strain DZ2, a Model Organism for Predation and Development.

Myxococcus xanthus is a member of the Myxococcales order within the Deltaproteobacteria subdivision. The myxobacteria reside in soil, have relatively large genomes, and display complex life cycles. Here, we report the whole-genome shotgun sequence of strain DZ2, which includes unique genes not found previously in strain DK1622.