C J Smith

Flavin mononucleotide (FMN)‐based fluorescent protein (FbFP) as reporter for gene expression in the anaerobe Bacteroides fragilis

In this study, we show the expression of flavin mononucleotide-based fluorescent protein (FbFP) BS2 as a marker for gene expression in the opportunistic human anaerobic pathogen Bacteroides fragilis. Bacteroides fragilis 638R strain carrying osu∷bs2 constructs showed inducible fluorescence following addition of maltose anaerobically compared with nonfluorescent cells under glucose-repressed conditions. Bacteria carrying ahpC∷bs2 or dps∷bs2 constructs were fluorescent following induction by oxygen compared with nonfluorescent cells from the anaerobic control cultures. In addition, when these transcriptional fusion constructs were mobilized into B. fragilis IB263, a constitutive peroxide response strain, fluorescent BS2, was detected in both anaerobic and aerobic cultures, confirming the unique properties of the FbFP BS2 to yield fluorescent signal in B. fragilis in the presence and in the absence of oxygen. Moreover, intracellular expression of BS2 was also detected when cell culture monolayers of J774.1 macrophages were incubated with B. fragilis ahpC∷bs2 or dps∷bs2 strains within an anaerobic chamber. This suggests that ahpC and dps are induced following internalization by macrophages. Thus, we show that BS2 is a suitable tool for the detection of gene expression in obligate anaerobic bacteria in in vivo studies.

The complex oxidative stress response of Bacteroides fragilis: the role of OxyR in control of gene expression

Gram-negative anaerobes in the genus Bacteroides are the predominant members of the GI-tract microflora where they play an important role in normal intestinal physiology. Bacteroides spp. also are significant opportunistic pathogens responsible for an array of intra-abdominal and other infections. Bacteroides fragilis is the most common anaerobic pathogen and it possesses virulence factors such as a capsule and neuraminidase that contribute to its success as a pathogen. Infection occurs when organisms escape from the anaerobic colon to aerobic sites such as the peritoneum where O(2) concentrations average 6%. Thus in addition to the classic virulence factors, resistance to oxidative stress is essential and may be involved in the initiation and persistence of infection. In fact, B. fragilis is highly O(2) tolerant, surviving extended periods (>24h) of O(2) exposure without a significant affect on viability. For protection against this oxidative stress B. fragilis mounts a complex physiological response that includes induction of >28 proteins involved in detoxification of oxygen radicals, protection of macromolecules, and adaptive physiology. One experimental strategy used to characterize this oxidative stress response is the direct detection of genes and proteins induced during exposure to O(2) or H(2)O(2). The methods employed have included RNA differential display to capture unique mRNA transcripts produced during oxidative stress, and native or 2D-gel electrophoresis to isolate and identify newly formed stress-induced proteins. Using these and other approaches a wide array of genes induced by oxidative stress have been discovered. These include genes for catalase, superoxide dismutase, thioredoxin-peroxidase, p20-peroxidase, cytochrome c peroxidase, Dps, alkyl hydroperoxidase, aerobic ribonucleotide reductase, ruberythrin, starch utilization, aspartate decarboxylase, and an RNA binding protein. The genes encoding these activities fall into three regulatory classes: (1) induced by O(2) only, (2) induced by H(2)O(2) only, and (3) induced by either O(2) or H(2)O(2). Such a complex regulatory response will likely involve multiple regulators. Thus far one regulator has been identified, OxyR, which controls a subset of the class 3 genes that are induced by either O(2) or H(2)O(2). OxyR responds rapidly to oxidative stress and transcriptional analyses have shown that OxyR-controlled genes are activated by as little as 0.5% O(2) or 10 microM H(2)O(2). Maximal expression of most OxyR regulon genes was reached at 50 microM H(2)O(2) and 2% O(2). These oxidant concentrations are similar to environmental levels that would be experienced by the organisms in tissues outside of the colon suggesting that the OxyR regulon would be induced during the course of an infection.

Characterization of the Bacteroides fragilis bfr Gene Product Identifies a Bacterial DPS-Like Protein and Suggests Evolutionary Links in the Ferritin Superfamily

A factor contributing to the pathogenicity of Bacteroides fragilis, the most common anaerobic species isolated from clinical infections, is the bacteriums extreme aerotolerance, which allows survival in oxygenated tissues prior to anaerobic abscess formation. We investigated the role of the bacterioferritin-related (bfr) gene in the B. fragilis oxidative stress response. The bfr mRNA levels are increased in stationary phase or in response to O(2) or iron. In addition, bfr null mutants exhibit reduced aerotolerance, and the bfr gene product protects DNA from hydroxyl radical cleavage in vitro. Crystallographic studies revealed a protein with a dodecameric structure and greater similarity to an archaeal DNA protection in starved cells (DPS)-like protein than to the 24-subunit bacterioferritins. Similarity to the DPS-like (DPSL) protein extends to the subunit and includes a pair of conserved cysteine residues juxtaposed to a buried dimetal binding site within the four-helix bundle. Compared to archaeal DPSLs, however, this bacterial DPSL protein contains several unique features, including a significantly different conformation in the C-terminal tail that alters the number and location of pores leading to the central cavity and a conserved metal binding site on the interior surface of the dodecamer. Combined, these characteristics confirm this new class of miniferritin in the bacterial domain, delineate the similarities and differences between bacterial DPSL proteins and their archaeal homologs, allow corrected annotations for B. fragilis bfr and other dpsl genes within the bacterial domain, and suggest an evolutionary link within the ferritin superfamily that connects dodecameric DPS to the (bacterio)ferritin 24-mer.

The Extracytoplasmic Function Sigma Factor EcfO Protects Bacteroides fragilis against Oxidative Stress

The anaerobe Bacteroides fragilis is a highly aerotolerant, opportunistic pathogen that is an important component of the human intestinal microbiota. Aerotolerance has been linked to a robust oxidative stress response, which in turn is necessary for maximal virulence in a mouse intra-abdominal abscess model. During oxidative stress, there is a dynamic change in gene expression that encompasses a third of the genome, but there is a paucity of information on factors that control this response. A large number of transcription regulators, including about 14 extracytoplasmic function (ECF) sigma factors, are affected by oxidative stress, and one of these, EcfO, was used as a model of ECF sigma factor activity during stress. Genetic and biochemical experiments showed that EcfO was located in an operon with a structurally unique anti-sigma factor, Reo. Cells deleted for EcfO were impaired during exposure to oxygen or other forms of oxidative stress, whereas reo mutants were more resistant to stress. Protein-protein interaction experiments demonstrated that Reo directly interacts with and regulates the activity of EcfO. Expression microarray and chromatin affinity precipitation assays were used to identify target genes regulated by EcfO, and an EcfO recognition sequence was identified. The results revealed that EcfO controls a regulon of novel lipoproteins whose distribution in nature is restricted to members of the Bacteroidetes phylum.

Genetic analysis of mobile tetQ elements in oral Prevotella species.

Prevotella species are members of the bacterial oral flora and are opportunistic pathogens in polymicrobial infections of soft tissues. Antibiotic resistance to tetracyclines is common in these bacteria, and the gene encoding this resistance has been previously identified as tetQ. The tetQ gene is also found on conjugative transposons in the intestinal Bacteroides species; whether these related bacteria have transmitted tetQ to Prevotella is unknown. In this study, we describe our genetic analysis of mobile tetQ elements in oral Prevotella species. Our results indicate that the mobile elements encoding tetQ in oral species are distinct from those found in the Bacteroides. The intestinal bacteria may act as a reservoir for the tetQ gene, but Prevotella has incorporated this gene into an IS21-family transposon. This transposon is present in Prevotella species from more than one geographical location, implying that the mechanism of tetQ spread between oral Prevotella species is highly conserved.

Bacteroides fragilis requires the ferrous-iron transporter FeoAB and the CobN-like proteins BtuS1 and BtuS2 for assimilation of iron released from heme

The intestinal commensal and opportunistic anaerobic pathogen Bacteroides fragilis has an essential requirement for both heme and free iron to support growth in extraintestinal infections. In the absence of free iron, B. fragilis can utilize heme as the sole source of iron. However, the mechanisms to remove iron from heme are not completely understood. In this study, we show that the inner membrane ferrous iron transporter ∆feoAB mutant strain is no longer able to grow with heme as the sole source of iron. Genetic complementation with the feoAB gene operon completely restored growth. Our data indicate that iron is removed from heme in the periplasmic space, and the released iron is transported by the FeoAB system. Interestingly, when B. fragilis utilizes iron from heme, it releases heme‐derived porphyrins by a dechelatase activity which is upregulated under low iron conditions. This is supported by the findings showing that formation of heme‐derived porphyrins in the ∆feoAB mutant and the parent strain increased 30‐fold and fivefold (respectively) under low iron conditions compared to iron replete conditions. Moreover, the btuS1 btuS2 double‐mutant strain (lacking the predicted periplasmic, membrane anchored CobN‐like proteins) also showed growth defect with heme as the sole source of iron, suggesting that BtuS1 and BtuS2 are involved in heme‐iron assimilation. Though the dechelatase mechanism remains uncharacterized, assays performed in bacterial crude extracts show that BtuS1 and BtuS2 affect the regulation of the dechelatase‐specific activities in an iron‐dependent manner. These findings suggest that the mechanism to extract iron from heme in Bacteroides requires a group of proteins, which spans the periplasmic space to make iron available for cellular functions.