Interactions of microbes and metals 12

Because these toxic metals are elements, they can not truly be broken down or destroyed, rather they can only be transferred to less toxic forms, sequestered away from cellular

processes to, or effluxed from, the cell (Bruins et al., 2000). Given that many of the

metals have unique properties, the mechanisms of resistance often differ. Resistance to some metals (lead and cadmium) is predominantly mediated by efflux transporters (Naik

and Dubey, 2013). Cadmium resistance is often mediated by the cadCA operon wherein

cadA is an ATP dependent cadmium efflux protein and cadC is a transcriptional repressor

(Bruins et al., 2000). Resistance to arsenic similarly involves the ars operon frequently

containing arsB (encoding an efflux transporter), arsC (encoding arsenate reductase) and

arsR (a transcriptional regulator)(Bruins et al., 2000). The ArsC enzyme is particularly

interesting as it converts arsenate to arsenite, a more toxic form of the metal, but one that is more easily transferred out of the cell (Rahman & Hassler, 2013).

In lactobacilli, toxic metals are often imported by nutrients acquisition systems, for example cadmium is imported through manganese transporters (Archibald and Duong, 1985). It is thought that cadmium sensitive organisms accumulate 3-15 more cadmium

than resistant counterparts (Trevors et al., 1986).

Mercury is unique among the toxic heavy metals in that mechanisms of resistance involve a true detoxification. Classical bacterial resistance to mercury involves the subsequent demethylation of organic mercury to inorganic mercury (organomercurial

lyase MerB), followed by the reduction of inorganic mercury to elemental mercury (mercuric reductase MerA) (Figure 1-1). While organic mercury can diffuse into the cell

due to its lipophilic nature, inorganic mercury (Hg2+) may be actively imported by the

MerTtransporter though this is variable among species (Bruins et al., 2000). The operon

is regulated by MerRwhich is a trans-acting repressor/activator protein which acts as a

repressor in the absence of mercury, but an activator its presence (Condee and Summers, 1992).

Figure 1-1. The Gram-positive mer operon of pI258.

In  the  mercury  resistance  operon  of  pI258  originally  identified  in  Staphylococcus  aureus,  the   gene  product  of  merT  facilitates  the  active  import  of  inorganic  mercury  while  the  products  of  

merA  and  merB  enzymatically  detoxify  mercury  from  organic  mercury  to  elemental  mercury   respectively.  The  operon  is  regulated  by  a  trans-­‐acting  repressor/activator  MerR.  

Much of the early study of metals focused on environmental bacteria. Much of the field of bacterial/metal interactions was pioneered by Simon Silver and his trainees (Silver, 2011). It wasn’t until 1972 that the nature of mercury resistance by volatilization of elemental mercury was discovered (Summers and Silver, 1972) and 1987 when the

genetic basis was uncovered (Laddaga et al., 1987). Since these early discoveries,

Cytosol Extracellular Environment

Hg2+ Hg2+ Hg2+ Hg2+ Hg2+ Hg0 NADPH NADP+ MerA Hg2+

merR merT merA merB

MerT MerR MerR Hg2+ MerB Hg -CH3 Hg2+ CH3 + Volatilization Sequestration

mercury resistance has been observed across a wide range of organisms including

Escherichia, Pseudomonas, Bacillus, Staphyloccocus, and Streptococcus. Despite being

present in a wide range of phylogenies, there has never been a published report of

mercury resistance elements present in species of Lactobacillus.

Not all biotransformations of mercury are favorable. It has long been known that certain

organisms such as Desulfovibrio and Geobacter are capable of converting inorganic

mercury to the more toxic methylmercury species (Schaefer et al., 2011). The mechanism

was not well understood until recently (Parks et al., 2013) where it was determined

through comparative genomics that a two-gene cluster (hgcA and hgcB) is sufficient and

capable of methylating mercury by acting as a corrinoid protein and a methyl

carrier/electron donor respectively. Furthermore, not all interactions of microbes and metals are enzymatically mediated, and passive electrostatic interactions with the cellular surface represent a major mechanism of interaction.

Early studies on interactions of bacterial cell wall used heavy metals as contrast agents in electronic microscopy (which was taken advantage of in Chapter 7 for imaging

purposes). Much of this work was carried out at Western University and the University of Guelph in the 1980s and greatly contributed to the understanding of mechanisms in this thesis (Beveridge and Murray, 1980; Beveridge and Koval, 1981; Beveridge and Murray,

1985, Beveridge, 1985; Mullen et al., 1989; Beveridge, 1989). Metals are normally found

in association with the membrane, peptidoglycan and teichoic acid of bacterial cells

where they have biological functions. For example, Mg2+ _{is essential for outer membrane}

integrity in E. coli. (Beveridge and Koval, 1981). While divalent calcium and magnesium

are usually the preferred metals on the cell surface, it is perhaps not surprising that heavy metals can also be found in association with the cell surface. Some years ago, it was noted that different cells have varying metal binding capacities reflecting variations in the cell wall’s chemistry, structure and constituent polymers. Lipopolysaccharide, for

example, contains phosphoryl groups that are thought to be a main site for metal

interactions in Gram-negative bacteria (Couglin et al., 1983). In Gram-positive cells, the

negatively charged phosphates of teichoic acid are the most likely repository of metals on the cell surface as extraction of teichoic acid from cells walls depletes most of the metal

content (Beveridge, 1989). Furthermore, additional cell surface features, such as the presence of capsule, may alter interactions. Capsule can block metals from interacting with the cell surface (Bitton and Frichofer, 1978) but may positively affect binding with

the cell depending on the chemical nature of the capsule (McLean et al., 1990). While

many studies have examined metal/microbe interactions for environmental

bioremediation, few have considered toxic metal interactions with members of the gut- microbiota.

Two studies have provided strong proof for a role of the gut microbiota in modulating

toxic metal uptake from the gut. Nakamura et al. (1977) showed that germ-free mice

lacking a gut microbiota absorbed considerably more mercury than their conventional

counterparts, and Breton et al. (2013) showed the same effect with both lead and

cadmium. While the germ-free mouse is a relatively extreme model for the role of a gut microbiota, more subtle microbiota modulating interventions have been tested using

antibiotics (Seko et al., 1981) and altered dietary fiber consumption (Rowland et al.,

1986). These have further implicated the gut microbiota in modulating host-mercury uptake. Little is known about mercury metabolism by the gut microbiota, but reference genomes available from the Human Microbiome Project, shotgun metagenomic

sequencing of fecal samples by the MetaHit consortium show evidence of both mercury detoxifying and methylating enzymes at the genetic level (unpublished observations, JE Bisanz).

Motivated by the results of studies on mercury uptake at different stages of early life

(Rowland et al., 1983), it was hypothesized that a gut microbiota enriched in

Bifidobacterium and Lactobacillus (prototypical probiotics) has a decreased ability to

favorably metabolize mercury due to the lack of mercury detoxifying enzymes normally found in these two genera. This puts an even greater emphasis on the need to develop probiotic strains with these traits.

While germ-free mouse models provided proof that the gut microbiota affects metal uptake from the gut, these early studies did not address an important potential mechanism independent of direct metal sequestration: modulation of host xenobiotic metabolism.