- Volume 163, Issue 12, 2017

The genomic integrity of Mycobacterium tuberculosis is continuously threatened by the harsh survival conditions inside host macrophages, due to immune and antibiotic stresses. Faithful genome maintenance and repair must be accomplished under stress for the bacillus to survive in the host, necessitating a robust DNA repair system. The importance of DNA repair systems in pathogenesis is well established. Previous examination of the M. tuberculosis genome revealed homologues of almost all the major DNA repair systems, i.e. nucleotide excision repair (NER), base excision repair (BER), homologous recombination (HR) and non-homologous end joining (NHEJ). However, recent developments in the field have pointed to the presence of novel proteins and pathways in mycobacteria. Homologues of archeal mismatch repair proteins were recently reported in mycobacteria, a pathway previously thought to be absent. RecBCD, the major nuclease-helicase enzymes involved in HR in E. coli, were implicated in the single-strand annealing (SSA) pathway. Novel roles of archeo-eukaryotic primase (AEP) polymerases, previously thought to be exclusive to NHEJ, have been reported in BER. Many new proteins with a probable role in DNA repair have also been discovered. It is now realized that the DNA repair systems in M. tuberculosis are highly evolved and have redundant backup mechanisms to mend the damage. This review is an attempt to summarize our current understanding of the DNA repair systems in M. tuberculosis.

The spirochete bacterium Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, the most common vector-borne disease in Europe and the United States. The spirochetes can be transmitted to humans via ticks, and then spread to different tissues, leading to arthritis, carditis and neuroborreliosis. Although antibiotics have commonly been used to treat infected individuals, some treated patients do not respond to antibiotics and experience persistent, long-term arthritis. Thus, there is a need to investigate alternative therapeutics against Lyme disease. The spirochete bacterium colonization is partly attributed to the binding of the bacterial outer-surface proteins to the glycosaminoglycan (GAG) chains of host proteoglycans. Blocking the binding of these proteins to GAGs is a potential strategy to prevent infection. In this review, we have summarized the recent reports of B. burgdorferi sensu lato GAG-binding proteins and discussed the potential use of synthetic and semi-synthetic compounds, including GAG analogues, to block pathogen interaction with GAGs. Such information should motivate the discovery and development of novel GAG analogues as new therapeutics for Lyme disease. New therapeutic approaches should eventually reduce the burden of Lyme disease and improve human health.

Herbicides are frequently released into both rural and urban environments. Commercial herbicide formulations induce adaptive changes in the way bacteria respond to antibiotics. Salmonella enterica sv. Typhimurium and Escherichia coli were exposed to common co-formulants of formulations, and S. enterica sv. Typhimurium was exposed to active ingredients dicamba, 2,4-D and glyphosate to determine what ingredients of the commercial formulations caused this effect. Co-formulants Tween80 and carboxymethyl cellulose induced changes in response, but the pattern of the responses differed from the active ingredients, and effect sizes were smaller. A commercial wetting agent did not affect antibiotic responses. Active ingredients induced changes in antibiotic responses similar to those caused by complete formulations. This occurred at or below recommended application concentrations. Targeted deletion of efflux pump genes largely neutralized the adaptive response in the cases of increased survival in antibiotics, indicating that the biochemistry of induced resistance was the same for formulations and specific ingredients. We found that glyphosate, dicamba, and 2,4-D, as well as co-formulants in commercial herbicides, induced a change in susceptibility of the potentially pathogenic bacteria E. coli and S. enterica to multiple antibiotics. This was measured using the efficiency of plating (EOP), the relative survival of the bacteria when exposed to herbicide and antibiotic, or just antibiotic, compared to survival on permissive media. This work will help to inform the use of non-medicinal chemical agents that induce changes in antibiotic responses.

S-adenosyl-l-methionine (AdoMet) is an essential metabolite, playing a wide variety of metabolic roles. The enzyme that produces AdoMet from l-methionine and ATP (methionine adenosyltransferase, MAT) is thus an attractive target for anti-cancer and antimicrobial agents. It would be very useful to have a system that allows rapid identification of species-specific inhibitors of this essential enzyme. A previously generated E. coli strain, lacking MAT (∆metK) but containing a heterologous AdoMet transporter, was successfully complemented with heterologous metK genes from several bacterial pathogens, as well as with MAT genes from a fungal pathogen and Homo sapiens. The nine tested genes, which vary in both sequence and kinetic properties, all complemented strain MOB1490 well in rich medium. When these strains were grown in glucose minimal medium, growth delays or defects were observed with some specific metK genes, defects that were dramatically reduced if l-methionine was added to the medium.

The fungal pathogen Batrachochytrium dendrobatidis has caused declines and extinctions in hundreds of amphibian species across the world. Virulence varies among and within lineages; the Global Panzootic Lineage (GPL) is the most pathogenic, although there is also variation in lethality among GPL isolates. Amphibians have a number of defences against pathogens, and skin products including the microbiota and host peptides have considerable influence over disease progression. Here we demonstrate that the collective skin products (the mucosome) of two amphibian species show significant variation in their ability to inhibit different globally distributed isolates of GPL. This may in part explain the variation in disease susceptibility of hosts to different strains of B. dendrobatidis. More work is required to identify particular traits associated with mucosomes that confer broad-spectrum inhibition across GPL in order to facilitate the development of prophylaxis and/or treatments for chytridiomycosis in situ.

Type VII secretion systems (T7SS) are found in many bacteria and secrete proteins involved in virulence and bacterial competition. In Staphylococcus aureus the small ubiquitin-like EsaB protein has been previously implicated as having a regulatory role in the production of the EsxC substrate. Here we show that in the S. aureus RN6390 strain, EsaB does not genetically regulate production of any T7 substrates or components, but is indispensable for secretion activity. Consistent with EsaB being an essential component of the T7SS, loss of either EsaB or EssC are associated with upregulation of a common set of iron acquisition genes. However, a further subset of genes were dysregulated only in the absence of EsaB. Quantitative western blotting indicates that EsaB is present at very low levels in cells. Substitution of a highly conserved threonine for alanine or arginine resulted in a loss of EsaB activity and destabilisation of the protein. Taken together our findings show that EsaB is essential for T7SS activity in RN6390.

Disulfide bonds confer stability and activity to proteins. Bioinformatic approaches allow predictions of which organisms make protein disulfide bonds and in which subcellular compartments disulfide bond formation takes place. Such an analysis, along with biochemical and protein structural data, suggests that many of the extremophile Crenarachaea make protein disulfide bonds in both the cytoplasm and the cell envelope. We have sought to determine the oxidative folding pathways in the sequenced genomes of the Crenarchaea, by seeking homologues of the enzymes known to be involved in disulfide bond formation in bacteria. Some Crenarchaea have two homologues of the cytoplasmic membrane protein VKOR, a protein required in many bacteria for the oxidation of bacterial DsbAs. We show that the two VKORs of Aeropyrum pernix assume opposite orientations in the cytoplasmic membrane, when expressed in E. coli. One has its active cysteines oriented toward the E. coli periplasm (ApVKORo) and the other toward the cytoplasm (ApVKORi). Furthermore, the ApVKORo promotes disulfide bond formation in the E. coli cell envelope, while the ApVKORi promotes disulfide bond formation in the E. coli cytoplasm via a co-expressed archaeal protein ApPDO. Amongst the VKORs from different archaeal species, the pairs of VKORs in each species are much more closely related to each other than to the VKORs of the other species. The results suggest two independent occurrences of the evolution of the two topologically inverted VKORs in archaea. Our results suggest a mechanistic basis for the formation of disulfide bonds in the cytoplasm of Crenarchaea.

Ralstonia pseudosolanacearum Ps29 showed repellent responses to alcohols including methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1,3-propanediol and prenol. R. pseudosolanacearum Ps29 possesses 22 putative chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). To identify a MCP involved in negative chemotaxis to ethanol, we measured ethanol chemotaxis of a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29. However, all the mutants showed repellent responses to ethanol comparable to that of the wild-type strain. We constructed a stepwise- and multiple-mcp gene deletion mutant collection of R. pseudosolanacearum Ps29. Analysis of the collection found that an 18-mcp-knockout mutant (strain POC18) failed to respond to ethanol. Complementation analysis using POC18 as the host strain found that introduction of mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 restored the ability of POC18 to respond to ethanol. However, unexpectedly, strain POC10II, harbouring unmarked deletions in 10 mcp genes including mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 showed repellent responses to ethanol comparable to that of wild-type Ps29. We hypothesised that multiple mcp mutations in POC18 led to a shortage of MCPs required for formation of functional chemoreceptor arrays. When pPS16 (encoding McpP involved in phosphate chemotaxis) was introduced into POC18, POC18(pPS16) did not respond to phosphate. This result supports the hypothesis. But, genetic analysis revealed that MCPs (Mcp07, Mcp13, Mcp20 and Mcp21) are not essential for ethanol chemotaxis. Thus, we conclude that many and unspecified MCPs are involved in negative chemotaxis to ethanol in R. pseudosolanacearum Ps29.

Bacterial O-antigens are synthesized on lipid carriers before being transferred to lipopolysaccharide core structures. Rhizobium etli CE3 lipopolysaccharide is a model for understanding O-antigen biological function. CE3 O-antigen structure and genetics are known. However, proposed enzymology for CE3 O-antigen synthesis has been examined very little in vitro, and even the sugar added to begin the synthesis is uncertain. A model based on mutagenesis studies predicts that 2-acetamido-2,6-dideoxy-d-glucose (QuiNAc) is the first O-antigen sugar and that genes wreV, wreQ and wreU direct QuiNAc synthesis and O-antigen initiation. Previously, synthesis of UDP-QuiNAc was shown to occur in vitro with a WreV orthologue (4,6-hexose dehydratase) and WreQ (4-reductase), but the WreQ catalysis in this conventional deoxyhexose-synthesis pathway was very slow. This seeming deficiency was explained in the present study after WreU transferase activity was examined in vitro. Results fit the prediction that WreU transfers sugar-1-phosphate to bactoprenyl phosphate (BpP) to initiate O-antigen synthesis. Interestingly, WreU demonstrated much higher activity using the product of the WreV catalysis [UDP-4-keto-6-deoxy-GlcNAc (UDP-KdgNAc)] as the sugar-phosphate donor than using UDP-QuiNAc. Furthermore, the WreQ catalysis with WreU-generated BpPP-KdgNAc as the substrate was orders of magnitude faster than with UDP-KdgNAc. The inferred product BpPP-QuiNAc reacted as an acceptor substrate in an in vitro assay for addition of the second O-antigen sugar, mannose. These results imply a novel pathway for 6-deoxyhexose synthesis that may be commonly utilized by bacteria when QuiNAc is the first sugar of a polysaccharide or oligosaccharide repeat unit: UDP-GlcNAc → UDP-KdgNAc → BpPP-KdgNAc → BpPP-QuiNAc.

The Escherichia coli PhoB-PhoR two-component system responds to phosphate starvation and induces the expression of many genes. Previous studies suggested that phosphate starvation induces oxidative stress, but the involvement of the PhoB regulon in oxidative stress tolerance has not been clarified. Here, we showed that ytfK, one of the PhoB regulon genes, is involved in cell tolerance to a redox-cycling drug, menadione, and H2O2 in stationary-phase cells. A ytfK deletion mutant was sensitive to H2O2 when the cells were grown anaerobically or micro-aerobically in the presence of nitrate. Genetic analysis suggested that the ytfK gene has a functional relationship with the oxyR and fur genes, among the oxyR regulon, at least, a catalase-encoding katG gene and peroxidase-encoding ahpCF genes. Overproduction of YtfK resulted in a KatG-dependent decrease of H2O2 concentration in the cell suspension, suggesting that katG is one of the targets of YtfK. Using a katG′-lacZ reporter fusion, we showed that YtfK enhances the transcription of katG although it was not clarified whether YtfK functions directly or not. We also showed that ytfK disruption results in reduced viability of stationary-phase cells under phosphate starvation. These results indicated that YtfK is involved in H2O2 tolerance by stimulating directly or indirectly the transcription of at least the catalase gene, and that this system plays an important role in cellular survival during phosphate starvation.

Small RNA (sRNA)-mediated regulation of gene expression is a major tool to understand bacterial responses to environmental changes. In particular, pathogenic bacteria employ sRNAs to adapt to the host environment and establish infection. Members of the Burkholderia cepacia complex, normally present in soil microbiota, cause nosocomial lung infection especially in hospitalized cystic fibrosis patients. We sequenced the draft genome of Burkholderia cenocepacia KC-01, isolated from the coastal saline soil, and identified several potential sRNAs in silico. Expression of seven small RNAs (Bc_KC_sr1–7) was subsequently confirmed. Two sRNAs (Bc_KC_sr1 and Bc_KC_sr2) were upregulated in response to iron depletion by 2,2’-bipyridyl and another two (Bc_KC_sr3 and Bc_KC_sr4) responded to the presence of 60 µM H2O2 in the culture media. Bc_Kc_sr5, 6 and 7 remained unchanged under these conditions. Expression of Bc_KC_sr2, 3 and 4 also altered with a change in temperature and incubation time. A search in the Rfam and BSRD databases identified Bc_Kc_sr4 as candidate738 in B. pseudomallei D286 and assigned Bc_Kc_sr5 and 6 as tmRNA and 6S RNA, respectively. The novel sRNAs were conserved in Burkholderiaceae but did not have any homologue in other genera. Bc_KC_sr1 and 4 were transcribed independently while the rest were part of the 3′ UTR of their upstream genes. TargetRNA2 predicted that these sRNAs could target a host of cellular messages with very high stringency. Intriguingly, regions surrounding the translation initiation site for several enzymes involved in Fe–S cluster and siderophore biosynthesis, ROS homeostasis, porins, transcription and translation regulators, were among the suggested putative binding sites for these sRNAs.

Ethylene is a gaseous signal sensed by plants and bacteria. Heterologous expression of the ethylene-forming enzyme (EFE) from Pseudomonas syringae in cyanobacteria leads to the production of ethylene under photoautotrophic conditions. The recent characterization of an ethylene-responsive signalling pathway affecting phototaxis in the cyanobacterium Synechocystis sp. PCC 6803 implied that biotechnologically relevant ethylene synthesis may induce regulatory processes that are not related to changes in metabolism. Here, we provide data that indicate that endogenously produced ethylene accelerates the movement of cells towards light. Microarray analysis demonstrates that ethylene mainly deactivates transcription from the csiR1/lsiR promoter, which is under the control of the two-component system consisting of the ethylene- and UV-A-sensing histidine kinase UirS and the DNA-binding response regulator UirR. Surprisingly, ethylene production triggers a very specific transcriptional response and only a few other smaller transcriptional changes are detected in the microarray analysis.