Multiple conformations facilitate PilT function in the type IV pilus.

Type IV pilus-like systems are protein complexes that polymerize pilin fibres. They are critical for virulence in many bacterial pathogens. Pilin polymerization and depolymerization are powered by motor ATPases of the PilT/VirB11-like family. This family is thought to operate with C2 symmetry; however, most of these ATPases crystallize with either C3 or C6 symmetric conformations. The relevance of these conformations is unclear. Here, we determine the X-ray structures of PilT in four unique conformations and use these structures to classify the conformation of available PilT/VirB11-like family member structures. Single particle electron cryomicroscopy (cryoEM) structures of PilT reveal condition-dependent preferences for C2, C3, and C6 conformations. The physiologic importance of these conformations is validated by coevolution analysis and functional studies of point mutants, identifying a rare gain-of-function mutation that favours the C2 conformation. With these data, we propose a comprehensive model of PilT function with broad implications for PilT/VirB11-like family members.

Real-time microscopy and physical perturbation of bacterial pili using maleimide-conjugated molecules.

Bacteria use surface-exposed, proteinaceous fibers called pili for diverse behaviors, including horizontal gene transfer, surface sensing, motility, and pathogenicity. Visualization of these filamentous nanomachines and their activity in live cells has proven challenging, largely due to their small size. Here, we describe a broadly applicable method for labeling and imaging pili and other surface-exposed nanomachines in live cells. This technique uses a combination of genetics and maleimide-based click chemistry in which a cysteine substitution is made in the major pilin subunit for subsequent labeling with thiol-reactive maleimide dyes. Large maleimide-conjugated molecules can also be used to physically interfere with the dynamic activity of filamentous nanomachines. We describe parameters for selecting cysteine substitution positions, optimized labeling conditions for epifluorescence imaging of pilus fibers, and methods for impeding pilus activity. After cysteine knock-in strains have been generated, this protocol can be completed within 30 min to a few hours, depending on the species and the experiment of choice. Visualization of extracellular nanomachines such as pili using this approach can provide a more comprehensive understanding of the role played by these structures in distinct bacterial behaviors.

Conformational Behaviour of Azasugars Based on Mannuronic Acid.

A set of mannuronic-acid-based iminosugars, consisting of the C-5-carboxylic acid, methyl ester and amide analogues of 1deoxymannorjirimicin (DMJ), was synthesised and their pH-dependent conformational behaviour was studied. Under acidic conditions the methyl ester and the carboxylic acid adopted an "inverted" 1 C4 chair conformation as opposed to the "normal" 4 C1 chair at basic pH. This conformational change is explained in terms of the stereoelectronic effects of the ring substituents and it parallels the behaviour of the mannuronic acid ester oxocarbenium ion. Because of this solution-phase behaviour, the mannuronic acid ester azasugar was examined as an inhibitor for a Caulobacter GH47 mannosidase that hydrolyses its substrates by way of a reaction itinerary that proceeds through a 3 H4 transition state. No binding was observed for the mannuronic acid ester azasugar, but sub-atomic resolution data were obtained for the DMJ⋅CkGH47 complex, showing two conformations-3 S1 and 1 C4 -for the DMJ inhibitor.

Conservation of the Essential Genome Among Caulobacter and Brevundimonas Species.

When the genomes of Caulobacter isolates NA1000 and K31 were compared, numerous genome rearrangements were observed. In contrast, similar comparisons of closely related species of other bacterial genera revealed nominal rearrangements. A phylogenetic analysis of the 16S rRNA indicated that K31 is more closely related to Caulobacter henricii CB4 than to other known Caulobacters. Therefore, we sequenced the CB4 genome and compared it to all of the available Caulobacter genomes to study genome rearrangements, discern the conservation of the NA1000 essential genome, and address concerns about using 16S rRNA to group Caulobacter species. We also sequenced the novel bacteria, Brevundimonas DS20, a representative of the genus most closely related to Caulobacter and used it as part of an outgroup for phylogenetic comparisons. We expected to find that there would be fewer rearrangements when comparing more closely related Caulobacters. However, we found that relatedness was not correlated with the amount of observed "genome scrambling." We also discovered that nearly all of the essential genes previously identified for C. crescentus are present in the other Caulobacter genomes and in the Brevundimonas genomes as well. However, a few of these essential genes were only found in NA1000, and some were missing in a combination of one or more species, while other proteins were 100 % identical across species. Also, phylogenetic comparisons of highly conserved genomic regions revealed clades similar to those identified by 16S rRNA-based phylogenies, verifying that 16S rRNA sequence comparisons are a valid method for grouping Caulobacters.

ClpXP and ClpAP proteolytic activity on divisome substrates is differentially regulated following the Caulobacter asymmetric cell division.

Proteolytic control of Caulobacter cell cycle proteins is primarily executed by ClpXP, a dynamically localized protease implicated in turnover of several factors critical for faithful cell cycle progression. Here, we show that the transient midcell localization of ClpXP that precedes cytokinesis requires the FtsZ component of the divisome. Although ClpAP does not exhibit subcellular localization, FtsZ is a substrate of both ClpXP and ClpAP in vivo and in vitro. A peptide containing the C-terminal portion of the FtsA divisome protein is a substrate of both ClpXP and ClpAP in vitro but is primarily degraded by ClpAP in vivo. Caulobacter carries out an asymmetric division in which FtsZ and FtsA are stable in stalked cells but degraded in the non-replicative swarmer cell where ClpAP alone degrades FtsA and both ClpAP and ClpXP degrade FtsZ. While asymmetric division in Caulobacter normally yields larger stalked and smaller swarmer daughters, we observe a loss of asymmetric size distribution among daughter cells when clpA is depleted from a strain in which FtsZ is constitutively produced. Taken together, these results suggest that the activity of both ClpXP and ClpAP on divisome substrates is differentially regulated in daughter cells.

Two independent spiral structures control cell shape in Caulobacter.

The actin homolog MreB contributes to bacterial cell shape. Here, we explore the role of the coexpressed MreC protein in Caulobacter and show that it forms a periplasmic spiral that is out of phase with the cytoplasmic MreB spiral. Both mreB and mreC are essential, and depletion of either protein results in a similar cell shape defect. MreB forms dynamic spirals in MreC-depleted cells, and MreC localizes helically in the presence of the MreB-inhibitor A22, indicating that each protein can form a spiral independently of the other. We show that the peptidoglycan transpeptidase Pbp2 also forms a helical pattern that partially colocalizes with MreC but not MreB. Perturbing either MreB (with A22) or MreC (with depletion) causes GFP-Pbp2 to mislocalize to the division plane, indicating that each is necessary but not sufficient to generate a helical Pbp2 pattern. We show that it is the division process that draws Pbp2 to midcell in the absence of MreB's regulation, because cells depleted of the tubulin homolog FtsZ maintain a helical Pbp2 localization in the presence of A22. By developing and employing a previously uncharacterized computational method for quantitating shape variance, we find that a FtsZ depletion can also partially rescue the A22-induced shape deformation. We conclude that MreB and MreC form spatially distinct and independently localized spirals and propose that MreB inhibits division plane localization of Pbp2, whereas MreC promotes lengthwise localization of Pbp2; together these two mechanism ensure a helical localization of Pbp2 and, thereby, the maintenance of proper cell morphology in Caulobacter.

Soft gel medium solidified with gellan gum for preliminary screening for root-associating, free-living nitrogen-fixing bacteria inhabiting the rhizoplane of plants.

For preliminary screening for and characterization of free-living nitrogen-fixing bacteria from rhizoplane microflora, we used Winogradsky's mineral mixture-based nitrogen-free medium solidified with 0.3% gellan gum. The soft gel medium enabled some reference and wild free-living nitrogen-fixing bacteria to grow in characteristic colonies, including their reaction to oxygen and their motility change. Gellan gum is thus likely to be a better gel matrix than agarose for the investigation of root-associating, free-living nitrogen-fixing bacteria to identify their characteristic behaviors.

Phylogeny and polyphasic taxonomy of Caulobacter species. Proposal of Maricaulis gen. nov. with Maricaulis maris (Poindexter) comb. nov. as the type species, and emended description of the genera Brevundimonas and Caulobacter.

The genus Caulobacter is composed of prosthecate bacteria often specialized for oligotrophic environments. The taxonomy of Caulobacter has relied primarily upon morphological criteria: a strain that visually appeared to be a member of the Caulobacter has generally been called one without challenge. A polyphasic approach, comprising 16S rDNA sequencing, profiling restriction fragments of 16S-23S rDNA interspacer regions, lipid analysis, immunological profiling and salt tolerance characterizations, was used to clarify the taxonomy of 76 strains of the genera Caulobacter. Brevundimonas, Hyphomonas and Mycoplana. The described species of the genus Caulobacter formed a paraphyletic group with Caulobacter henricii, Caulobacter fusiformis, Caulobacter vibrioides and Mycoplana segnis (Caulobacter segnis comb. nov.) belonging to Caulobacter sensu stricto. Caulobacter bacteroides (Brevundimonas bacteroides comb. nov.), C. henricii subsp. aurantiacus (Brevundimonas aurantiaca comb. nov.), Caulobacter intermedius (Brevundimonas intermedia comb. nov.), Caulobacter subvibrioides (Brevundimonas subvibrioides comb. nov.), C. subvibrioides subsp. albus (Brevundimonas alba comb. nov.), Caulobacter variabilis (Brevundimonas variabilis comb. nov.) and Mycoplana bullata belong to the genus Brevundimonas. The halophilic species Caulobacter maris and Caulobacter halobacteroides are different from these two genera and form the genus Maricaulis gen. nov. with Maricaulis maris as the type species. Caulobacter leidyia was observed to cluster with species of the genus Sphingomonas. Caulobacter crescentus is synonymous with C. vibrioides and C. halobacteroides is synonymous with Maricaulis maris as determined by these analyses and DNA-DNA hybridization. Biomarkers discerning these different genera were determined. The necessary recombinations have been proposed and a description of Maricaulis is presented.

Unusual lipid composition of the gram-negative, freshwater, stalked bacterium Caulobacter bacteroides NP-105.

The extractable lipids of the gram-negative, stalked, freshwater bacterium Caulobacter bacteroides NP-105 account for about 9.5% by weight of dry cells, polar lipids comprising up to 95% of the total. The polar lipids consist of five glycolipids, namely, 1,2-diacyl-3-alpha-D-glucopyranosyl-sn-glycerol (I) (34% of the total), 1,2-diacyl-3-alpha-D-[6'-(1",2"-diacyl-sn-glycero-3-phospho)]glucopyranosyl-sn-glycerol (II) (7%), 1,2-diacyl-3-a-D-glucuronopyranosyl-sn-glycerol (III) (17%), 1,2-diacyl-3-alpha-D-(6'-sulfo)quinovopyranosyl-sn-glycerol (V) (9%), and 1,2-diacyl-3-alpha-D-[4'-(alpha-D-glucopyranosyl)] glucuronopyranosyl-sn-glycerol (VI) (28%), and one glycerophospholipid, 1,2-diacyl-sn-glycero-3-phosphoglycerol (IV) (5%). The main fatty acyls of the lipids are n-16:0, cis-18:1 omega7, and cis-11,12-methyleneoctadecanoic (lactobacillic) acid residue. Of the 6 lipids listed, only 3 (I, IV, and V) can be treated as widely encountered. However, sulfonic glycolipid V is a characteristic lipid component of photosynthetic organisms rather than non-photosynthetic ones. Phosphatidyl derivatives of glycosyldiacylglycerols of type II normally occur in gram-positive bacteria, among gram-negative bacteria they have been revealed only in two species of the Pseudomonas genus. Glucuronosyl and alpha-glucosyl-a-glucuronosyl diacylglycerols such as III and VI, respectively, are very rare lipids, the latter being found so far only in a Streptomyces strain.

Reassessment of the phylogenetic position of Caulobacter subvibrioides.

Determination of the 16S rRNA gene sequence of Caulobacter subvibrioides ATCC 15264T (T = type strain) confirmed that this species is a member of the alpha subclass of the Proteobacteria and showed that it is phylogenetically most closely related to the Caulobacter group comprising the species Caulobacter bacteroides, Caulobacter crescentus, and Brevundimonas (Pseudomonas) diminuta, for which 16S rRNA sequences of the type strains are currently available. The closest known relative of strain ATCC 15264T among these species is B. diminuta (level of direct pairwise sequence similarity, 95%). On the basis of its previously determined 16S rRNA sequence (accession number M83797), C. subvibrioides is most closely related to Sphingomonas adhaesiva in the alpha-4 subgroup (level of similarity, 97.7%). Analysis of the hydroxy fatty acids of C. subvibrioides ATCC 15264T showed that the 2-hydroxymyristic acid which is characteristic of the genus Sphingomonas was absent.

Phylogeny of Prosthecobacter, the fusiform caulobacters: members of a recently discovered division of the bacteria.

Prosthecobacter fusiformis is morphologically similar to caulobacters; however, it lacks a dimorphic life cycle. To determine the relatedness of the genus Prosthecobacter to dimorphic caulobacters and other prosthecate members of the alpha subgroup of the Proteobacteria (alpha-Proteobacteria), we isolated and sequenced 16S rRNA genes from four Prosthecobacter strains. Surprisingly, the results of phylogenetic analyses placed the fusiform caulobacters in a deeply rooted division of the Bacteria that was most closely affiliated with the Planctomyces-Chlamydia group and only distantly related to the alpha-Proteobacteria. The genus Prosthecobacter shares a common lineage in this division with Verrucomicrobium spinosum, a polyprosthecate, heterotrophic bacterium. Consistent with this phylogenetic placement, menaquinones were isolated from Prosthecobacter strains and menaquinones have been isolated from Verrucomicrobium strains and planctomycetes but not from members of the alpha-Proteobacteria. Thus, the genus Prosthecobacter is a second genus in the recently described order Verrucomicrobiales. Members of the genus Prosthecobacter are susceptible to beta-lactam antibiotics and contain mesodiaminopimelic acid, indicating that they, unlike members of the Planctomycetales or Chlamydiales, have peptidoglycan cell walls. This major phenotypic difference, together with the phylogenetic independence of the verrucomicrobia, indicates that these bacteria and the sources of related 16S ribosomal DNAs obtained from soils, freshwater, and the marine pelagic environment represent an unrecognized division of the Bacteria.

Isolation and comparison of the paracrystalline surface layer proteins of freshwater caulobacters.

Several methods for isolation of the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A were evaluated. Treatment of cells with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 2 was the most effective means of selectively removing RsaA from cells, and after neutralization, the protein was capable of reassembling into a paracrystalline structure. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid treatment could also be used to extract RsaA and yielded protein capable of reassembly. The success of the methods was likely related to disruption of calcium-mediated bonding; calcium was required for recrystallization, while magnesium and strontium ions were ineffective. Antibody was raised against purified RsaA and, along with the S-layer extraction techniques, was used to evaluate 42 strains of caulobacters isolated from a variety of aquatic and wastewater treatment locations. A single characteristic protein could be isolated from the 35 strains that produced an S layer; with one exception, no proteins were extracted from strains that had no S layer. The presumed S-layer proteins ranged in size from 100 to 193 kDa. All of these proteins specifically reacted with anti-RsaA serum by Western immunoblot analysis. In strain CB15A, a specific S-layer-associated oligosaccharide has been proposed to be involved in a calcium-mediated attachment of the S layer to the cell surface. This molecule was detected by Western immunoblotting with a specific antiserum and on polyacrylamide gels stained for polysaccharides. A comparable band was found in all S-layer-producing strains and for most, S-layer-associated oligosaccharide-specific antibody reacted with them in Western analysis. Overall, in freshwater caulobacters at least portions of their S-layer structures appear to be strongly conserved entities, as well as the means of attachment to the cell surface.