Susan F. Koval

V. Functions of S‐layers

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

Advances in bacterial paracrystalline surface layers

Introduction: A Perspective on SLayer Research (R.G.E. Murray). Structural Analysis of SLayers: SLayers Found on Clinical Isolates (K. Lounatmaa et al.). SLayers of Agricultural and Environmental Importance: Predation on Bacteria Possessing SLayers (S.F. Koval). Chemistry and Molecular Biology of SLayers: Glycoprotein Nature of Select Bacterial SLayers (P. Messner et al.). SLayer Glycoproteins from Moderately and Extremely Halophilic Archaeobacteria (M. Sumper). Applications for SLayers: Molecular Nanotechnology with SLayers (S. Pum, U.B. Sleytr). Stable Liposomes Formed from Archaeal Ether Lipids (C.G. Choquet et al.). Poster Presentations: Linker Mutagenesis of the Caulobacter crescentus SLayer Protein (W.H. Bingle et al.). Summary Statements: Summary Statements (T.J. Beveridge et al.). 28 additional articles. Index.

Isolation and characterization of novel alkaliphiles from bauxite-processing waste and description of Bacillus vedderi sp. nov., a new obligate alkaliphile

Summary Three novel alkaliphilic bacteria were isolated and characterized following enrichment at pH 10, using mud from a bauxite-processing red mud tailing pond as source material. The isolates, designated strains JaA, JaD and JaH were aerobic, Gram-positive, spore-forming, mesophilic rods which were motile by means of peritrichously arranged flagella. Strains JaD and JaH could also grow anaerobically. All three isolates had pH optima for growth of approximately 10, with strains JaD and JaH obligate allcaliphiles. Strains JaA and JaD grew in the presence of 10% (w/v) NaCl while strain JaH could grow in the presence of up to 7.5% (w/v) NaCl. The mole %G+C content of purified DNA was 41.3% for strain JaA, 41.8% for strain JaD and 38.3% for strain JaH. The new isolates appear to be members of the genus Bacillus but 16s rDNA sequence analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis comparisons of whole cell protein profiles, as well as nutritional, biochemical and morphological traits indicated the new isolates are not members of any validly published Bacillus species. The epithet Bacillus vedderi is proposed for strain JaH.

The Isolation and Characterization of Lipopolysaccharide-defective Mutants of Pseudomonas aeruginosa PAC1

Mutants with defective lipopolysaccharides (LPSs) were isolated from Pseudomonas aeruginosa PACIR (Habs serogroup 3) by selection for resistance to aeruginocin from P. aeruginosa PI6 Carbenicillin-sensitive mutants were isolated from P. aeruginosa PACI but not all had defective LPSs. Rough colonial morphology and resistance to bacteriophage II9X appeared to be independent of LPS composition. The LPSs from five mutants were analysed and compared with that of the parent strain. Separation of partially-degraded polysaccharides from LPS from PACI on Sephadex G75 yielded two different high molecular weight fractions and a phosphorylated low molecular weight fraction (L). The mutant LPSs lacked most or all of the high molecular weight fractions but retained some low molecular weight material. That from PACI and two of the mutants was separated by elution from Biogel P6 into two fractions. One, L2, was the core polysaccharide while the other, LI, contained short antigenic side-chains attached to the core like the semi-rough (SR) LPSs of the Enterobacteriaceae. The two mutants which gave the LI fraction with Habs 3 and PACI antisera as did the parent strain. The other three mutants were unreactive and their LPSs contained core components only. One appeared to have a complete core while the other two lacked rhamnose and rhammose plus glucose respectively. Thus there may be four types of LPS in PACI: one contains unsubstituted core polysaccharide and yields L2 on acid hydrolysis, another has short antigenic side-chains of the SR type and yields the LI fraction, while the two high molecular weight fractions are derived from core polysaccharides with different side-chains.

Bacterial capsules: no barrier against Bdellovibrio.

Bdellovibrio bacteriovorus 109J attached to both capsulated and non-capsulated Escherichia coli K29 cells. Electron microscopy revealed penetration of the thick polysaccharide capsule without any major disintegration of the neighbouring capsular matrix. The capsule remained intact during bdelloplast formation and lysis was unaffected by capsulation of the prey cell. This study shows that, in contrast to its effect on bacteriophage penetration and its protective activities against immune defence mechanisms, the capsule of E. coli does not serve as a barrier against invasion by B. bacteriovorus.

Bdellovibrio exovorus sp. nov., a novel predator of Caulobacter crescentus

The life cycle, prey range and taxonomic status of a Bdellovibrio-like organism, strain JSS(T), were studied. Strain JSS(T) was isolated from sewage in London, Ontario, Canada, in enrichment culture with Caulobacter crescentus prey cells. During predation, this strain remained attached to the outside of a stalked C. crescentus cell. No periplasmic growth stage was observed and no bdelloplast was formed. The stalked cells of C. crescentus retained their shape and, after predation, were devoid of cytoplasmic content, as shown by transmission electron microscopy. A periplasmic growth stage has been a definitive character in the description of members of the genera Bdellovibrio, Bacteriovorax, Bacteriolyticum and Peredibacter. This is the first description of an epibiotic predator in this group of prokaryotic predators. The G+C content of the genomic DNA of strain JSS(T) was 46.1 mol%. 16S rRNA gene sequence analysis showed that this strain was related to Bdellovibrio bacteriovorus strains HD100(T), 109J, 114 and 127 (90-93 % similarity). Phylogenetic analysis based on 16S rRNA gene sequences grouped strain JSS(T) with the Bdellovibrio cluster, but at a distance from other Bdellovibrio isolates. On the basis of features of the life cycle and phylogenetic data, it was concluded that strain JSS(T) merits classification as the type strain of a novel species, for which the name Bdellovibrio exovorus sp. nov. is proposed (type strain JSS(T) =ATCC BAA-2330(T) = DSM 25223(T)).

Characterization of type IV pili in the life cycle of the predator bacterium Bdellovibrio

Bdellovibrio and like organisms (BALOs) are obligate prokaryotic predators of other Gram-negative bacteria. Bdellovibrio bacteriovorus is the most studied organism among BALOs. It has a periplasmic life cycle with two major stages: a motile, non-replicative stage spent searching for prey (the attack phase) and a stage spent inside the periplasm of the Gram-negative prey cell (the growth phase) after forming an osmotically stable body termed the bdelloplast. Within Bdellovibrio, there are also strains exhibiting an epibiotic life cycle. The genome sequence of the type strain B. bacteriovorus HD100(T) revealed the presence of multiple dispersed pil genes encoding type IV pili. Type IV pili in other bacteria are involved in adherence to and invasion of host cells and therefore can be considered to play a role in invasion of prey cells by Bdellovibrio. In this study, genes involved in producing type IV pili were identified in the periplasmic strain B. bacteriovorus 109J and an epibiotic Bdellovibrio sp. strain JSS. The presence of fibres on attack-phase cells was confirmed by examining negative stains of cells fixed with 10% buffered formalin. Fibres were at the non-flagellated pole on approximately 25% of attack-phase cells. To confirm that these fibres were type IV pili, a truncated form of PilA lacking the first 35 amino acids was designed to facilitate purification of the protein. The truncated PilA fused to a His-tag was overexpressed in Escherichia coli BL21(DE3) plysS. The fusion protein, accumulated in the insoluble fraction, was purified under denaturing conditions and used to produce polyclonal antisera. Immunoelectron microscopy showed that polar fibres present on the cell surface of the predator were composed of PilA, the major subunit of type IV pili. Immunofluorescence microscopy showed the presence of pilin on attack-phase cells of B. bacteriovorus 109J during attachment to prey cells and just after penetration, inside the bdelloplast. Antibodies against PilA delayed and inhibited predation in co-cultures of Bdellovibrio. This study confirms that type IV pili play a role in invasion of prey cells by Bdellovibrio.

In and out: an analysis of epibiotic vs periplasmic bacterial predators

Bdellovibrio and like organisms (BALO) are obligate predators of Gram-negative bacteria, belonging to the α- and δ-proteobacteria. BALO prey using either a periplasmic or an epibiotic predatory strategy, but the genetic background underlying these phenotypes is not known. Here we compare the epibiotic Bdellovibrio exovorus and Micavibrio aeruginosavorus to the periplasmic B. bacteriovorus and Bacteriovorax marinus. Electron microscopy showed that M. aeruginosavorus, but not B. exovorus, can attach to prey cells in a non-polar manner through its longitudinal side. Both these predators were resistant to a surprisingly high number of antibiotic compounds, possibly via 26 and 19 antibiotic-resistance genes, respectively, most of them encoding efflux pumps. Comparative genomic analysis of all the BALOs revealed that epibiotic predators have a much smaller genome (ca. 2.5 Mbp) than the periplasmic predators (ca. 3.5 Mbp). Additionally, periplasmic predators have, on average, 888 more proteins, at least 60% more peptidases, and one more rRNA operon. Fifteen and 219 protein families were specific to the epibiotic and the periplasmic predators, respectively, the latter clearly forming the core of the periplasmic ‘predatome’, which is upregulated during the growth phase. Metabolic deficiencies of epibiotic genomes include the synthesis of inosine, riboflavin, vitamin B6 and the siderophore aerobactin. The phylogeny of the epibiotic predators suggests that they evolved by convergent evolution, with M. aeruginosavorus originating from a non-predatory ancestor while B. exovorus evolved from periplasmic predators by gene loss.

Relatedness of the flagellins from methanogens

Purified flagellar filaments isolated from six methanogens were composed of multiple flagellins. Two flagellins were present in Methanococcus deltae (Mr=34000 and 32000), Methanoculleus marisnigri (Mr=31000 and 25500) and Methanococcus jannaschii (Mr=31000 and 27500), three in Methanothermus fervidus (Mr=34000, 25000 and 24000) and four or more in both Methanococcus vanniellii and Methanococcus maripaludis (Mrranging from 27500 to 32000). The flagellins of M. fervidus and M. deltae reacted positively with glycoprotein-specific stains. The flagellins of M. deltae, M. maripaludis and M. vannielii were closely related to those of M. voltae based on cross-reactivity with antisera raised against M. voltae flagellins and homology with flagellin-specific oligonucleotide probes to the N-terminus and leader peptide of M. voltae flagellins. Similarities appear to exist among the flagellins of M. fervidus, M. marisnigri and Halobacterium halobium based on cross-reactivity with antisera produced against the flagella of Methanospirillum hungatei JF1. The N-termini of the flagellins from the mesophilic Methanococcus spp. and M. marisnigri show homology with the N-termini of other archaebacterial flagellins. These N-termini may undergo a modification involving removal of a leader peptide.

Mutation of a Broadly Conserved Operon (RL3499-RL3502) from Rhizobium leguminosarum Biovar viciae Causes Defects in Cell Morphology and Envelope Integrity

The bacterial cell envelope is of critical importance to the function and survival of the cell; it acts as a barrier against harmful toxins while allowing the flow of nutrients into the cell. It also serves as a point of physical contact between a bacterial cell and its host. Hence, the cell envelope of Rhizobium leguminosarum is critical to cell survival under both free-living and symbiotic conditions. Transposon mutagenesis of R. leguminosarum strain 3841 followed by a screen to isolate mutants with defective cell envelopes led to the identification of a novel conserved operon (RL3499-RL3502) consisting of a putative moxR-like AAA(+) ATPase, a hypothetical protein with a domain of unknown function (designated domain of unknown function 58), and two hypothetical transmembrane proteins. Mutation of genes within this operon resulted in increased sensitivity to membrane-disruptive agents such as detergents, hydrophobic antibiotics, and alkaline pH. On minimal media, the mutants retain their rod shape but are roughly 3 times larger than the wild type. On media containing glycine or peptides such as yeast extract, the mutants form large, distorted spheres and are incapable of sustained growth under these culture conditions. Expression of the operon is maximal during the stationary phase of growth and is reduced in a chvG mutant, indicating a role for this sensor kinase in regulation of the operon. Our findings provide the first functional insight into these genes of unknown function, suggesting a possible role in cell envelope development in Rhizobium leguminosarum. Given the broad conservation of these genes among the Alphaproteobacteria, the results of this study may also provide insight into the physiological role of these genes in other Alphaproteobacteria, including the animal pathogen Brucella.