Genome sequences of four agarolytic bacteria from the Bacteroidia and Gammaproteobacteria.

Here we present the genomes of four marine agarolytic bacteria belonging to the Bacteroidota and Proteobacteria. Two genomes are closed and two are in draft form, but all are at least 99% complete and offer new opportunities to study agar-degradation in marine bacteria.

Recovered microbiome of an oviparous lizard differs across gut and reproductive tissues, cloacal swabs, and faeces.

Microbial diversity and community function are related, and can be highly specialized in different gut regions. The cloacal microbiome of Sceloporus virgatus females provides antifungal protection to eggshells, a specialized function that suggests a specialized microbiome. Here, we describe the cloacal, intestinal, and oviductal microbiome from S. virgatus gravid females, adding to growing evidence of microbiome localization in reptiles and other taxa. We further assessed whether common methods for sampling gastrointestinal (GI) microbes - cloacal swabs and faeces - provide accurate representations of these microbial communities. We found that different regions of the gut had unique microbial communities. The cloacal microbiome showed extreme specialization averaging 99% Proteobacteria (Phylum) and 83% Enterobacteriacaea (Family). Enterobacteriacaea decreased up the GI and reproductive tracts. Cloacal swabs recovered communities similar to that of lower intestine and cloacal tissues. In contrast, faecal samples had much higher diversity and a distinct composition (common Phyla: 62% Firmicutes, 18% Bacteroidetes, 10% Proteobacteria; common families: 39% Lachnospiraceae, 11% Ruminococcaceae, 11% Bacteroidaceae) relative to all gut regions. The common families in faecal samples made up <1% of cloacal tissue samples, increasing to 43% at the upper intestine. Similarly, the common families in gut tissue (Enterobacteriaceae and Helicobacteraceae) made up <1% of the faecal microbiome. Further, we found that cloacal swabs taken shortly after defaecation may be contaminated with faecal matter. Our results serve as a caution against using faeces as a proxy for GI microbes, and may help explain high between-sample variation seen in some studies using cloacal swabs.

Complete Genome Sequence of the Predatory Bacterium Ensifer adhaerens Casida A.

We report here the complete genome sequence of the facultative predatory bacterium Ensifer adhaerens strain Casida A. The genome was assembled into three circular contigs, with a main chromosome as well as two large secondary replicons, that totaled 7,267,502 bp with 6,641 predicted open reading frames.

Atomic force microscopy of bacterial communities.

This chapter discusses atomic force microscopy (AFM) for the benefit of microbiologists who are interested in using this technique to examine the structures and dynamics of bacteria. AFM is a powerful technique for imaging biological samples at the nanometer to micrometer scale under nondestructive conditions. In order to be imaged with AFM, bacteria must be supported by a surface, which presents challenges because many laboratory strains of bacteria are planktonic. Still, in nature many bacteria live at surfaces and interfaces. This chapter discusses the benefits and difficulties of different methods that have been used to support bacteria on surfaces for AFM imaging and presents two methods in detail used to successfully grow and image bacteria at solid-liquid and solid-air interfaces. Using these methods it is possible to study bacterial morphology and interactions in a native state. These explorations by AFM have important applications to the study of different kinds of bacteria, interfacial bacterial communities, and biofilms.

Predation, death, and survival in a biofilm: Bdellovibrio investigated by atomic force microscopy.

Biofilms are complex microbial communities that are resistant to attack by bacteriophages and to removal by drugs and chemicals. Here we use atomic force microscopy (AFM) to image the attack on Escherichia coli biofilms by Bdellovibrio bacteriovorus 109J. Bdellovibrio is a small, predatory bacterium that invades and devours other Gram-negative bacteria. We demonstrate that under dilute nutrient conditions, bdellovibrios can prevent the formation of simple bacterial biofilms and destroy established biofilms; under richer conditions the prey bacteria persist and are not eradicated, but may be shifted toward solution populations. Using AFM we explore these bacterial interactions with more detail and accuracy than available by more traditional staining assays or optical microscopy. AFM also allows us to investigate the nanoscale morphological changes of the predator, especially those related to motility. This demonstration of Bdellovibrio's successful predation in a biofilm inspires us to consider ways that it might be used productively for industrial, medical, agricultural, and biodefensive purposes.

Characterization of pES213, a small mobilizable plasmid from Vibrio fischeri.

Most Vibrio fischeri strains isolated from the Euprymna scolopes light organ carry plasmids, often including both a large (>40kb) plasmid, and one or more small (<12kb) plasmids. The large plasmids share homology with pES100, which is the lone plasmid in V. fischeri type strain ES114. pES100 appears to encode a conjugative system similar to that on plasmid R721. The small plasmids lack extensive similarity to pES100, but they almost always occur in cells that also harbor a large plasmid resembling pES100. We found that many or all of these small plasmids share homology with pES213, a plasmid in strain ES213. We determined the 5501-bp pES213 sequence and generated selectable antibiotic resistance encoding pES213 derivatives, which enabled us to examine replication, retention, and transfer in V. fischeri. An 863-bp fragment of pES213 with features characteristic of theta-type replicons conferred replication without requiring any pES213 open reading frame (ORF). We estimated that pES213 derivatives were maintained at 9.4 copies per genome, which corresponds well with a model of random plasmid segregation to daughter cells and the approximately 10(-4) per generation frequency of plasmid loss. pES213 derivatives mobilized between V. fischeri strains at frequencies up to approximately 10(-4) in culture and in the host, apparently by employing the pES100 conjugative apparatus. pES213 carries two homologs of the putative pES100 origin of transfer (oriT), and V. fischeri strains lacking the pES100 conjugative relaxase, including a relaxase mutant, failed to serve as donors for transmission of pES213 derivatives. In other systems, genes directing conjugative transfer can function in trans to oriT, so it was noteworthy that ORFs adjacent to oriT, VFB51 in pES100 and traYZ in pES213, enhanced transfer 100- to 1000-fold when provided in cis. We also identified and disrupted the V. fischeri recA gene. RecA was not required for stable pES213 replication but surprisingly was required in donors for efficient transfer of pES213 derivatives. These studies provide an explanation for the prevalence and co-occurrence of pES100- and pES213-type plasmids, illuminate novel elements of pES213 mobilization, and provide the foundation for new genetic tools in V. fischeri.

Predatory prokaryotes: an emerging research opportunity.

Predatory prokaryotes have evolved a unique strategy of obtaining energy and biosynthetic materials from their surroundings: acquiring them from other living bacterial cells. These types of microbes have been found in a diverse variety of environments, and may play an important role in modulating microbial population structure and dynamics, as has been hypothesized for marine viruses and possibly protists. Only one genus of predatory bacterium, Bdellovibrio, has been extensively described and studied, though several other examples have been reported in the literature. In this review, the four basic strategies used by currently described predatory prokaryotes will be discussed: "wolfpack" group predation, epibiotic attachment, direct cytoplasmic invasion, and periplasmic invasion. Special adaptations to each approach will be considered, and compared overall to the genetic and biochemical characteristics of symbiotic or pathogenic prokaryotes living within eukaryotic cells. Two specific examples of predatory microbes, Bdellovibrio and Ensifer, will be described in terms of predation strategy, association with host cells, and host range. The prospects for bringing to bear the tools of molecular microbial genetics to the study of predatory prokaryotes will be explored, using current research with Bdellovibrio and Ensifer as examples.

Investigations into the life cycle of the bacterial predator Bdellovibrio bacteriovorus 109J at an interface by atomic force microscopy.

Atomic force microscopy was used to image Bdellovibrio bacteriovorus 109J, a gram-negative bacterial predator that consumes a variety of other gram-negative bacteria. In predator-prey communities grown on filters at hydrated air-solid interfaces, repeated cycles of hunting, invasion, growth, and lysis occurred readily even though the cells were limited to near two-dimensional movement. This system allowed us to image the bacteria directly without extensive preparation or modification, and many of the cells remained alive during imaging. Presented are images of the life cycle in two species of prey organisms, both Escherichia coli (a small prey bacterium that grows two-dimensionally on a surface) and Aquaspirillum serpens (a large prey bacterium that grows three-dimensionally on a surface), including high-resolution images of invaded prey cells called bdelloplasts. We obtained evidence for multiple invasions per prey cell, as well as significant heterogeneity in morphology of bdellovibrios. Mutant host-independent bdellovibrios were observed to have flagella and to excrete a coating that causes the predators to clump together on a surface. Most interestingly, changes in the texture of the cell surface membranes were measured during the course of the invasion cycle. Thus, coupled with our preparation method, atomic force microscopy allowed new observations to be made about Bdellovibrio at an interface. These studies raise important questions about the ways in which bacterial predation at interfaces (air-solid or liquid-solid) may be similar to or different from predation in solution.