- Volume 163, Issue 11, 2017

Bacillus licheniformis strains are used for the large-scale production of industrial exoenzymes from proteinaceous substrates, but details of the amino acid metabolism involved are largely unknown. In this study, two chromosomal genes putatively involved in amino acid metabolism of B. licheniformis were deleted to clarify their role. For this, a convenient counterselection system for markerless in-frame deletions was developed for B. licheniformis. A deletion plasmid containing up- and downstream DNA segments of the chromosomal deletion target was conjugated to B. licheniformis and integrated into the genome by homologous recombination. Thereafter, the counterselection was done by using a codBA cassette. The presence of cytosine deaminase and cytosine permease exerted a conditionally lethal phenotype on B. licheniformis cells in the presence of the cytosine analogue 5-fluorocytosine. Thereby clones were selected that lost the integrated vector sequence and the anticipated deletion target after a second recombination step. This method allows the construction of markerless mutants in Bacillus strains in iterative cycles. B. licheniformis MW3 derivatives lacking either one of the ORFs BL03009 or BL00190, encoding a putative alanine dehydrogenase and a similar putative enzyme, respectively, retained the ability to grow in minimal medium supplemented with alanine as the carbon source. In the double deletion mutant MW3 ΔBL03009 ΔBL00190, however, growth on alanine was completely abolished. These data indicate that the two encoded enzymes are paralogues fulfilling mutually replaceable functions in alanine utilization, and suggest that in B. licheniformis MW3 alanine utilization is initiated by direct oxidative transamination to pyruvate and ammonium.

Clinicians often have to deal with infections that are difficult to control because they are caused by superbugs resistant to many antibiotics. Alternatives to antibiotic treatment include antimicrobial photodynamic therapy (aPDT). The photodynamic process causes bacterial death, inducing oxidative stress through the photoactivation of photosensitizer molecules in the presence of oxygen. No PDT-resistant bacteria have been selected to date, thus the response to photo-oxidative stress in non-phototrophic bacteria needs further investigation. The opportunistic pathogen Pseudomonas aeruginosa, in particular, has been shown to be more tolerant to PDT than other micro-organisms. In order to find any genetic determinants involved in PDT-tolerance, a panel of transposon mutants of P. aeruginosa PAO1 involved in the quorum sensing signalling system and membrane cytoplasmic transport were photoinactivated as part of this study. Two pseudomonas quinolone signalling (PQS) knock-out mutants, pqsH - and pqsC -, were as PDT-sensitive as the PAO1 wild-type strains. Two PQS hyperproducer variants, pqsA - and rsaL -, were shown to be more tolerant to photo-oxidative stress than the wild-type strain. In the pqsA - mutant, the hyperpigmentation due to the presence of phenazines could protect cells against PDT stress, while in rsaL - no pigmentation was detectable. Furthermore, a mutant impaired in an ATP-binding cassette transport involved in maintaining the asymmetry of the outer membrane was significantly more tolerant to photo-oxidative stress than the wild-type strain. These observations support the involvement of quorum sensing and the importance of the bacterial cell envelope when dealing with photo-oxidative stress induced by photodynamic treatment.

Stenotrophomonas maltophilia, a Gram-negative, multi-drug-resistant bacterium, is increasingly recognized as a key opportunistic pathogen. Thus, we embarked upon an investigation of S. maltophilia iron acquisition. To begin, we determined that the genome of strain K279a is predicted to encode a complete siderophore system, including a biosynthesis pathway, an outer-membrane receptor for ferrisiderophore, and other import and export machinery. Compatible with these data, K279a and other clinical isolates of S. maltophilia secreted a siderophore-like activity when grown at 25–37 °C in low-iron media, as demonstrated by a chrome azurol S assay, which detects iron chelation, and Arnow and Rioux assays, which detect catecholate structures. Importantly, these supernatants rescued the growth of iron-starved S. maltophilia, documenting the presence of a biologically active siderophore. A mutation in one of the predicted biosynthesis genes (entC) abolished production of the siderophore and impaired bacterial growth in low-iron conditions. Inactivation of the putative receptor gene (fepA) prevented the utilization of siderophore-containing supernatants for growth in low-iron conditions. Although the biosynthesis and import loci showed some similarity to those of enterobactin, a well-known catecholate made by enteric bacteria, the siderophore of K279a was unable to rescue the growth of an enterobactin-utilizing indicator strain, and conversely iron-starved S. maltophilia could not use purified enterobactin. Furthermore, the S. maltophilia siderophore displayed patterns of solubility in organic compounds and mobility upon thin-layer chromatography that were distinct from those of enterobactin and its derivative, salmochelin. Together, these data demonstrate that S. maltophilia secretes a novel catecholate siderophore.

Overuse of antibiotics is contributing to an emerging antimicrobial resistance crisis. To better understand how bacteria adapt tolerance and resist antibiotic treatment, Pseudomonas aeruginosa isolates obtained from infection sites sampled from companion animals were collected and evaluated for phenotypic differences. Selected pairs of clonal isolates were obtained from individual infection samples and were assessed for antibiotic susceptibility, cyclic di-GMP levels, biofilm production, motility and genetic-relatedness. A total of 18 samples from equine, feline and canine origin were characterized. A sample from canine otitis media produced a phenotypically heterogeneous pair of P. aeruginosa isolates, 42121A and 42121B, which during growth on culture medium respectively exhibited hyper dye-binding small colony morphology and wild-type phenotypes. Antibiotic susceptibility to gentamicin and ciprofloxacin also differed between this pair of clonal isolates. Sequence analysis of gyrA, a gene known to be involved in ciprofloxacin resistance, indicated that 42121A and 42121B both contained mutations that confer ciprofloxacin resistance, but this did not explain the differences in ciprofloxacin resistance that were observed. Cyclic di-GMP levels also varied between this pair of isolates and were shown to contribute to the observed colony morphology variation and ability to form a biofilm. Our results demonstrate the role of cyclic di-GMP in generating the observed morphological phenotypes that are known to contribute to biofilm-mediated antibiotic tolerance. The generation of phenotypic diversity may go unnoticed during standard diagnostic evaluation, which potentially impacts the therapeutic strategy chosen to treat the corresponding infection and may contribute to the spread of antibiotic resistance.

Acinetobacter baumannii is a ubiquitous multidrug-resistant bacteria that is found on a variety of surfaces, including skin, hair and soil. During the past decade, A. baumannii has emerged as a significant cause of nosocomial infections in the United States. Recent studies have highlighted the ability of some bacteria to utilize a wide variety of fatty acids as a membrane remodelling strategy. Considering this, we hypothesized that fatty acids may have an effect on the emerging pathogen A. baumannii. Thin-layer chromatography indicated structural alterations to major phospholipids. Liquid chromatography/mass spectrometry confirmed the assimilation of numerous exogenous polyunsaturated fatty acids (PUFAs) into the phospholipid species of A. baumannii. The incorporation of fatty acids affected several bacterial phenotypes, including membrane permeability, biofilm formation, surface motility and antimicrobial peptide resistance.

Genus Comamonas is a group of bacteria that are able to degrade a variety of environmental waste. Comamonas aquatica CJG (C. aquatica) in this genus is able to absorb low-density lipoprotein but not high-density lipoprotein of human serum. Using 1H and 13C NMR spectroscopy, we found that the O-polysaccharide (O-antigen) of this bacterium is comprised of a disaccharide repeat (O-unit) of d-glucose and 2-O-acetyl-l-rhamnose, which is shared by Serratia marcescens O6. The O-antigen gene cluster of C. aquatica, which is located between coaX and tnp4 genes, contains rhamnose synthesis genes, glycosyl and acetyl transferase genes, and ATP-binding cassette transporter genes, and therefore is consistent with the O-antigen structure determined here.

Verticillins are the dimeric epipolythiodioxopiperazines (ETPs) produced by the fungus Clonostachys rogersoniana. Despite their profound biological effects, they are commonly produced in rice medium as complex mixtures that are difficult to separate, limiting further study and evaluation for this class of metabolites. Therefore, there is an urgent need to understand the regulation of verticillin biosynthesis. Recently, we cloned the biosynthetic gene cluster of verticillin (ver), and identified the only regulatory gene verZ in this cluster. The deduced product of verZ contains a basic Zn(II)2Cys6 DNA-binding domain. Disruption of verZ significantly reduced the production of 11′-deoxyverticillin A (C42) and decreased the transcriptional level of the verticillin biosynthetic genes. To further reveal its function, a recombinant gene encoding the DNA-binding domain of VerZ was expressed in E. coli and the His6-tagged VerZbd was purified to homogeneity by Ni-NTA chromatography. Electrophoretic mobility shift assays (EMSAs) showed that VerZbd bound specifically to the promoter regions of the verticillin biosynthetic genes. Bioinformatic analysis of the VerZbd-binding regions revealed a conserved palindromic sequence of (T/C)(C/A)(G/T)GN3CC(G/T)(A/G)(G/C). Base substitution of the conserved sequence completely abolished the binding activity of VerZbd to its targets. These results suggested that VerZ controls verticillin production through directly activating transcription of the biosynthetic genes in C. rogersoniana.

During conditions of nutrient limitation bacteria undergo a series of global gene expression changes to survive conditions of amino acid and fatty acid starvation. Rapid reallocation of cellular resources is brought about by gene expression changes coordinated by the signalling nucleotides' guanosine tetraphosphate or pentaphosphate, collectively termed (p)ppGpp and is known as the stringent response. The stringent response has been implicated in bacterial virulence, with elevated (p)ppGpp levels being associated with increased virulence gene expression. This has been observed in the highly pathogenic Francisella tularensis sub spp. tularensis SCHU S4, the causative agent of tularaemia. Here, we aimed to artificially induce the stringent response by culturing F. tularensis in the presence of the amino acid analogue l-serine hydroxamate. Serine hydroxamate competitively inhibits tRNAser aminoacylation, causing an accumulation of uncharged tRNA. The uncharged tRNA enters the A site on the translating bacterial ribosome and causes ribosome stalling, in turn stimulating the production of (p)ppGpp and activation of the stringent response. Using the essential virulence gene iglC, which is encoded on the Francisella pathogenicity island (FPI) as a marker of active stringent response, we optimized the culture conditions required for the investigation of virulence gene expression under conditions of nutrient limitation. We subsequently used whole genome RNA-seq to show how F. tularensis alters gene expression on a global scale during active stringent response. Key findings included up-regulation of genes involved in virulence, stress responses and metabolism, and down-regulation of genes involved in metabolite transport and cell division. F. tularensis is a highly virulent intracellular pathogen capable of causing debilitating or fatal disease at extremely low infectious doses. However, virulence mechanisms are still poorly understood. The stringent response is widely recognized as a diverse and complex bacterial stress response implicated in virulence. This work describes the global gene expression profile of F. tularensis SCHU S4 under active stringent response for the first time. Herein we provide evidence for an association of active stringent response with FPI virulence gene expression. Our results further the understanding of the molecular basis of virulence and regulation thereof in F. tularensis. These results also support research into genes involved in (p)ppGpp production and polyphosphate biosynthesis and their applicability as targets for novel antimicrobials.

The switch from a motile, planktonic existence to an attached biofilm is a major bacterial lifestyle transition that is often mediated by complex regulatory pathways. In this report, we describe a CheY-like protein required for control of the motile-to-sessile switch in the plant pathogen Agrobacterium tumefaciens. This regulator, which we have designated ClaR, possesses two distinct CheY-like receiver (REC) domains and is involved in the negative regulation of biofilm formation, through production of the unipolar polysaccharide (UPP) adhesin and cellulose. The ClaR REC domains share predicted structural homology with characterized REC domains and contain the majority of active site residues known to be essential for protein phosphorylation. REC1 is missing the conserved aspartate (N72) residue and although present in REC 2 (D193), it is not required for ClaR-dependent regulation suggesting that phosphorylation, which modulates the activity of many CheY-like proteins, appears not to be essential for ClaR activity. We also show that ClaR-dependent negative regulation of attachment is diminished significantly in mutants for PruA and PruR, proteins known to be involved in a pterin-mediated attachment regulation pathway. In A. tumefaciens, pterins are required for control of the intracellular signal cyclic diguanylate monophosphate through the DcpA regulator, but our findings suggest that pterin-dependent ClaR control of attachment can function independently from DcpA, including dampening of c-di-GMP levels. This report of a novel CheY-type biofilm regulator in A. tumefaciens thus also adds significant details to the role of pterin-mediated signalling.

Yeast cells can use γ-aminobutyric acid (GABA), a non-protein amino acid, as a nitrogen source that is mainly imported by the permease Uga4 and catabolized by the enzymes GABA transaminase and succinate-semialdehyde dehydrogenase, encoded by the UGA1 and UGA2 genes, respectively. The three UGA genes are inducible by GABA and subject to nitrogen catabolite repression. Hence, their regulation occurs through two mechanisms, one dependent on the inducer and the other on nitrogen source quality. The aim of this work was to better understand the molecular mechanisms of transcription factors acting on different regulatory elements present in UGA promoters, such as Uga3, Dal81, Leu3 and the GATA factors, and to establish the mechanism of the concerted action between them. We found that Gat1 plays an important role in the induction of UGA4 transcription by GABA and that Gzf3 has an effect in cells grown in a poor nitrogen source such as proline and that this effect is positive on UGA4 expression. We also found that Gln3 and Dal80 affect the interaction of Uga3 and Dal81 on UGA promoters. Moreover, our results indicated that the repressing activity of Leu3 on UGA4 and UGA1 occurs through Dal80 since we demonstrated that Leu3 facilitates Dal80 interaction with DNA. However, when the expression of GATA factors is null or negligible, Leu3 functions as an activator.

N-Lysine acetylation is a dynamic, reversible and regulatory post-translational modification (PTM) in prokaryotes, which integrates and coordinates metabolisms responding to environmental clues. However, the molecular mechanism underlying the signalling pathway from nutrient sensing to protein acetylation remains incompletely understood in micro-organisms. Here we found that global nitrogen regulator GlnR directly controls transcription of genes encoding lysine deacetylases in Actinobacteria. Electrophoretic mobility shift assays and real-time PCR (RT-PCR) in three Actinobacteria species (Saccharopolyspora erythraea, Streptomyces coelicolor and Mycobacterium smegmatis) revealed that GlnR regulator protein is able to interact with the promoter regions of these genes and activate their transcription. Furthermore, it was demonstrated that cellular acetylation status (acetylome) is modulated by extracellular nitrogen availability. Our results present an example of the novel complete signal transduction mechanism of regulating protein deacetylation through a nutrient-sensing pleiotropic regulator in response to nutrient availability.

Cyanobacteria acclimatize to nitrogen deprivation by changing cellular metabolism. The nitrogen-regulated response regulator A (NrrA) is involved in regulation of carbon metabolism in response to nitrogen deprivation. However, it has not been elucidated whether these regulatory functions of NrrA are particular to a few model strains or are general among diverse cyanobacteria. In this study, we showed that regulation and functions of NrrA were highly conserved among β-cyanobacteria, which included physiologically and ecologically diverse strains. All β-cyanobacteria had the nrrA gene, while it was absent in α-cyanobacteria. The canonical NtcA-dependent promoter sequence was found upstream of the nrrA genes in most β-cyanobacteria, and its expression was indeed induced by nitrogen deprivation. Biochemical and physiological analyses of NrrA from phylogenetically distinct cyanobacteria indicated that regulation of NrrA activity and NrrA functions, namely activation of glycogen catabolism, were also common to β-cyanobacteria. These results support the conclusion that NrrA plays an important role in acclimatization to nitrogen deprivation, and that activation of glycogen catabolism is a primitive response to nitrogen deprivation in β-cyanobacteria.

Neisseria gonorrhoeae is the causative agent of gonorrhoea, the second most common bacterial sexually transmitted disease. Riboregulation mediated by small regulatory RNAs (sRNAs) is increasingly recognized as an important means of gene expression control in this human-restricted pathogen. sRNAs act at the post-transcriptional level by base-pairing with their target mRNAs which affects translation initiation and/or mRNA stability. In this study we initiated the characterization of a pair of highly conserved sRNAs of N. gonorrhoeae which exhibit redundant functions in the control of a common set of target genes. The identified targets of the sibling sRNAs NgncR_162 and NgncR_163 participate in basic metabolic processes including the methylcitrate and citrate cycle, aa uptake and degradation, and also in transcription regulation. Our data indicate that the sibling sRNAs control their targets via direct base-pairing between the same single-stranded domain(s) of the sRNA and the ribosome binding site in the 5′-untranslated region of the mRNA.