Robert A. Britton

Identification of Catabolite Repression as a Physiological Regulator of Biofilm Formation by Bacillus subtilis by Use of DNA Microarrays

Biofilms are structured communities of cells that are encased in a self-produced polymeric matrix and are adherent to a surface. Many biofilms have a significant impact in medical and industrial settings. The model gram-positive bacterium Bacillus subtilis has recently been shown to form biofilms. To gain insight into the genes involved in biofilm formation by this bacterium, we used DNA microarrays representing >99% of the annotated B. subtilis open reading frames to follow the temporal changes in gene expression that occurred as cells transitioned from a planktonic to a biofilm state. We identified 519 genes that were differentially expressed at one or more time points as cells transitioned to a biofilm. Approximately 6% of the genes of B. subtilis were differentially expressed at a time when 98% of the cells in the population were in a biofilm. These genes were involved in motility, phage-related functions, and metabolism. By comparing the genes differentially expressed during biofilm formation with those identified in other genomewide transcriptional-profiling studies, we were able to identify several transcription factors whose activities appeared to be altered during the transition from a planktonic state to a biofilm. Two of these transcription factors were Spo0A and sigma-H, which had previously been shown to affect biofilm formation by B. subtilis. A third signal that appeared to be affecting gene expression during biofilm formation was glucose depletion. Through quantitative biofilm assays and confocal scanning laser microscopy, we observed that glucose inhibited biofilm formation through the catabolite control protein CcpA.

Histamine derived from probiotic Lactobacillus reuteri suppresses TNF via modulation of PKA and ERK signaling.

Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s) produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC) separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA), histidine/histamine antiporter (hdcP), and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2)-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H2 receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA) and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases.

Role of the Intestinal Microbiota in Resistance to Colonization by Clostridium difficile

Antibiotic-associated infection with the bacterial pathogen Clostridium difficile is a major cause of morbidity and increased health care costs. C difficile infection follows disruption of the indigenous gut microbiota by antibiotics. Antibiotics create an environment within the intestine that promotes C difficile spore germination, vegetative growth, and toxin production, leading to epithelial damage and colitis. Studies of patients with C difficile infection and animal models have shown that the indigenous microbiota can inhibit expansion and persistence of C difficile. Although the specific mechanisms of these processes are not known, they are likely to interfere with key aspects of the pathogens physiology, including spore germination and competitive growth. Increasing our understanding of how the intestinal microbiota manage C difficile could lead to better means of controlling this important nosocomial pathogen.

Host-microbial symbiosis in the vertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm

Vertebrates engage in symbiotic associations with vast and complex microbial communities that colonize their gastrointestinal tracts. Recent advances have provided mechanistic insight into the important contributions of the gut microbiome to vertebrate biology, but questions remain about the evolutionary processes that have shaped symbiotic interactions in the gut and the consequences that arise for both the microbes and the host. Here we discuss the biological principles that underlie microbial symbiosis in the vertebrate gut and the potential of the development of mutualism. We then review phylogenetic and experimental studies on the vertebrate symbiont Lactobacillus reuteri that have provided novel insight into the ecological and evolutionary strategy of a gut microbe and its relationship with the host. We argue that a mechanistic understanding of the microbial symbiosis in the vertebrate gut and its evolution will be important to determine how this relationship can go awry, and it may reveal possibilities by which the gut microbiome can be manipulated to support health.

Interaction between the intestinal microbiota and host in Clostridium difficile colonization resistance

Clostridium difficile infection (CDI) has become one of the most prevalent and costly nosocomial infections. In spite of the importance of CDI, our knowledge of the pathogenesis of this infection is still rudimentary. Although previous use of antibiotics is generally considered to be the sine qua non of CDI, the mechanisms by which antibiotics render the host susceptible to C. difficile are not well defined. In this review, we will explore what is known about how the indigenous microbiota acts in concert with the host to prevent colonization and virulence of C. difficile and how antibiotic administration disturbs host-microbiota homeostasis, leading to CDI.

Maturation of the 5' end of Bacillus subtilis 16s rRNA by the essential ribonuclease YkqC/RNase J1

Functional ribosomal RNAs are generated from longer precursor species in every organism known. Maturation of the 5′ side of 16S rRNA in Escherichia coli is catalysed in a two‐step process by the cooperative action of RNase E and RNase G. However, many bacteria lack RNase E and RNase G orthologues, raising the question as to how 16S rRNA processing occurs in these organisms. Here we show that the maturation of Bacillus subtilis 16S rRNA is also a two‐step process and that the enzyme responsible for the generation of the mature 5′ end is the widely distributed essential ribonuclease YkqC/RNase J1. Depletion of B. subtilis of RNase J1 results in an accumulation of 16S rRNA precursors in vivo. The precursor species are found in polysomes suggesting that they can function in translation. Mutation of the predicted catalytic site of RNase J1 abolishes both 16S rRNA processing and cell viability. Finally, purified RNase J1 can correctly mature precursor 16S rRNA assembled in 70S ribosomes, showing that its role is direct.

Cell cycle arrest in Era GTPase mutants: a potential growth rate‐regulated checkpoint in Escherichia coli

Era is a low‐molecular‐weight GTPase essential for Escherichia coli viability. The gene encoding Era is found in the rnc operon, and the synthesis of both RNase III and Era increases with growth rate. Mutants that are partially defective in Era GTPase activity or that are reduced in the synthesis of wild‐type Era become arrested in the cell cycle at the predivisional two‐cell stage. The partially defective Era GTPase mutation (era1) suppresses several temperature‐sensitive lethal alleles that affect chromosome replication and chromosome partitioning but not cell division. Our results suggest that Era plays an important role in cell cycle progression at a specific point in the cycle, after chromosome partitioning but before cytokinesis. Possible functions for Era in cell cycle progression and the initiation of cell division are discussed.

Genomic and genetic characterization of the bile stress response of probiotic Lactobacillus reuteri ATCC 55730

ABSTRACT Probiotic bacteria encounter various stresses after ingestion by the host, including exposure to the low pH in the stomach and bile in the small intestine. The probiotic microorganism Lactobacillus reuteri ATCC 55730 has previously been shown to survive in the human small intestine. To address how L. reuteri can resist bile stress, we performed microarray experiments to determine gene expression changes that occur when the organism is exposed to physiological concentrations of bile. A wide variety of genes that displayed differential expression in the presence of bile indicated that the cells were dealing with several types of stress, including cell envelope stress, protein denaturation, and DNA damage. Mutations in three genes were found to decrease the strains ability to survive bile exposure: lr1864, a Clp chaperone; lr0085, a gene of unknown function; and lr1516, a putative esterase. Mutations in two genes that form an operon, lr1584 (a multidrug resistance transporter in the major facilitator superfamily) and lr1582 (unknown function), were found to impair the strains ability to restart growth in the presence of bile. This study provides insight into the possible mechanisms that L. reuteri ATCC 55730 may use to survive and grow in the presence of bile in the small intestine.

Probiotic L. reuteri Treatment Prevents Bone Loss in a Menopausal Ovariectomized Mouse Model

Estrogen deficiency is a major risk factor for osteoporosis that is associated with bone inflammation and resorption. Half of women over the age of 50 will experience an osteoporosis related fracture in their lifetime, thus novel therapies are needed to combat post‐menopausal bone loss. Recent studies suggest an important role for gut‐bone signaling pathways and the microbiota in regulating bone health. Given that the bacterium Lactobacillus reuteri ATCC PTA 6475 (L. reuteri) secretes beneficial immunomodulatory factors, we examined if this candidate probiotic could reduce bone loss associated with estrogen deficiency in an ovariectomized (Ovx) mouse menopausal model. Strikingly, L. reuteri treatment significantly protected Ovx mice from bone loss. Osteoclast bone resorption markers and activators (Trap5 and RANKL) as well as osteoclastogenesis are significantly decreased in L. reuteri‐treated mice. Consistent with this, L. reuteri suppressed Ovx‐induced increases in bone marrow CD4+ T‐lymphocytes (which promote osteoclastogenesis) and directly suppressed osteoclastogenesis in vitro. We also identified that L. reuteri treatment modifies microbial communities in the Ovx mouse gut. Together, our studies demonstrate that L. reuteri treatment suppresses bone resorption and loss associated with estrogen deficiency. Thus, L. reuteri treatment may be a straightforward and cost‐effective approach to reduce post‐menopausal bone loss. J. Cell. Physiol. 229: 1822–1830, 2014.

The antimicrobial compound reuterin (3-hydroxypropionaldehyde) induces oxidative stress via interaction with thiol groups.

Reuterin is an antimicrobial compound produced by Lactobacillus reuteri, and has been proposed to mediate, in part, the probiotic health benefits ascribed to this micro-organism. Despite 20 years of investigation, the mechanism of action by which reuterin exerts its antimicrobial effects has remained elusive. Here we provide evidence that reuterin induces oxidative stress in cells, most likely by modifying thiol groups in proteins and small molecules. Escherichia coli cells subjected to sublethal levels of reuterin expressed a set of genes that overlapped with the set of genes composing the OxyR regulon, which senses and responds to various forms of oxidative stress. E. coli cells mutated for oxyR were more sensitive to reuterin compared with wild-type cells, further supporting a role for reuterin in exerting oxidative stress. The addition of cysteine to E. coli or Clostridium difficile growth media prior to exposure to reuterin suppressed the antimicrobial effect of reuterin on these bacteria. Interestingly, interaction with E. coli stimulated reuterin production or secretion by L. reuteri, indicating that contact with other microbes in the gut increases reuterin output. Thus, reuterin inhibits bacterial growth by modifying thiol groups, which indicates that reuterin negatively affects a large number of cellular targets.