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Microbiology Society logo - Volume 165, Issue 1, 2019

Volume 165, Issue 1, 2019

  • Microbe Profile

      • Coxiella burnetii: A Pathogenic Intracellular Acidophile

        Coxiella burnetii is an obligate intracellular pathogen that causes acute and chronic Q fever. C. burnetii grows within a eukaryotic host cell in a vacuole highly similar to a phagolysosome. Found worldwide, this environmentally stable pathogen is maintained in nature via chronic infection of ruminants. Aerosol-mediated infection of humans results in infection and usurpation of alveolar macrophages through mechanisms using a bacterial Type 4B Secretion System and secreted effector proteins. Advances in axenic culture and genetic systems are changing our understanding of the pathogen’s physiology and intimate molecular manipulations of host cells during infection.

  • Biotechnology

      • Increasing lipid production using an NADP+-dependent malic enzyme from Rhodococcus jostii

        The occurrence of NADP+-dependent malic enzymes (NADP+-MEs) in several Rhodococcus strains was analysed. The NADP+-ME number in Rhodococcus genomes seemed to be a strain-dependent property. Total NADP+-ME activity increased by 1.8- and 2.6-fold in the oleaginous Rhodococcus jostii RHA1 and Rhodococcus opacus PD630 strains during cultivation under nitrogen-limiting conditions. Total NADP+-ME activity inhibition by sesamol resulted in a significant decrease of the cellular biomass and lipid production in oleaginous rhodococci. A non-redundant ME coded by the RHA1_RS44255 gene located in a megaplasmid (pRHL3) of R. jostii RHA1 was characterized and its heterologous expression in Escherichia coli resulted in a twofold increase in ME activity in an NADP+-dependent manner. The overexpression of RHA1_RS44255 in RHA1 and PD630 strains grown on glucose promoted an increase in total NADP+-ME activity and an up to 1.9-foldincrease in total fatty acid production without sacrificing cellular biomass. On the other hand, its expression in Rhodococcus fascians F7 grown on glycerol resulted in a 1.3–1.4-foldincrease in total fatty acid content. The results of this study confirmed the contribution of NADP+-MEs to TAG accumulation in oleaginous rhodococci and the utility of these enzymes as an alternative approach to increase bacterial oil production from different carbon sources.

  • Host-Microbe Interaction

      • SrcA is a chaperone for the Salmonella SPI-2 type three secretion system effector SteD

        Effector proteins of type three secretion systems (T3SS) often require cytosolic chaperones for their stabilization, to interact with the secretion machinery and to enable effector delivery into host cells. We found that deletion of srcA, previously shown to encode a chaperone for the Salmonella pathogenicity island 2 (SPI-2) T3SS effectors SseL and PipB2, prevented the reduction of mature Major Histocompatibility Complex class II (mMHCII) from the surface of antigen-presenting cells during Salmonella infection. This activity was shown previously to be caused by the SPI-2 T3SS effector SteD. Since srcA and steD are located in the same operon on the Salmonella chromosome, this suggested that the srcA phenotype might be due to an indirect effect on SteD. We found that SrcA is not translocated by the SPI-2 T3SS but interacts directly and forms a stable complex with SteD in bacteria with a 2 : 1 stoichiometry. We found that SrcA was not required for SPI-2 T3SS-dependent, neutral pH-induced secretion of either SseL or PipB2 but was essential for secretion of SteD. SrcA therefore functions as a chaperone for SteD, explaining its requirement for the reduction in surface levels of mMHCII.

  • Physiology and Metabolism

    • Avoiding amino acid depletion in a complex medium results in improved Escherichia coli BW25113 growth

      We studied Escherichia coli BW25113 growth in a complex medium with emphasis on amino acid consumption. The aim was to profile amino acid utilization in acid-hydrolysed casein and a defined nutrient-rich medium and based on these measurements modify the medium for better growth performance. Amino acid depletions in both media caused apparent biomass growth stops that prolonged growth duration. Obtained amino acid consumption values enabled a new defined medium to be formulated, where no growth stops were observed, the specific growth rate was constant, and the provided substrates were fully utilized. Similarly, we modified the acid-hydrolysed casein medium by adding pure amino acids that removed the apparent biomass growth stops. Key to our results was the combination of growth medium analysis and process monitoring data, specifically oxygen partial pressure and produced carbon dioxide that were used to track growth changes. Our findings showed the deficiencies of the nutrient-rich medium and how rational medium design, based on consumption values, removed these shortcomings. The resulting balanced medium gives a high specific growth rate and is suitable for studying E. coli physiology at fast growth.

    • The quantity and distribution of biofilm growth of Escherichia coli strain ATCC 9723 depends on the carbon/energy source

      Escherichia coli strain 15 (ATCC 9723) formed robust biofilms of two distinct forms on glass tubes. In rich, low-osmolarity medium, the biofilms were restricted to the air/liquid interface, resulting in rings attached to the glass. As it was not evident that these biofilms extended across the liquid surface, we termed them ‘ring’ rather than ‘pellicle’ biofilms. In minimal medium supplemented with a non-fermentable substrate as the carbon/energy source, we observed either robust ring biofilms or little biofilm of any type, depending on the substrate. In contrast, fermentable substrates (sugars and sugar derivatives) supported robust biofilms covering most of the solid/liquid interface, which we termed ‘tube-covering biofilms’. Maximal biofilm growth was observed when the sugar was a relatively poor substrate, supporting slow growth and known to cause minimal dephosphorylation of regulatory protein Enzyme IIAGlucose of the phosphotransferase system. Compounds found to be inhibitors of biofilm growth, such as lactate, caused a shift from tube-covering to ring form at low concentration and complete loss of biofilm growth at high when added to minimal medium supplemented with a fermentable substrate. Exogenous cAMP activated biofilm growth under all conditions tested, leading to more intense ring or tube-covering biofilms and/or to a shift from ring to tube-covering form.

  • Regulation

      • Dynamic changes in lysine acetylation and succinylation of the elongation factor Tu in Bacillus subtilis

        N ε-lysine acetylation and succinylation are ubiquitous post-translational modifications in eukaryotes and bacteria. In the present study, we showed a dynamic change in acetylation and succinylation of TufA, the translation elongation factor Tu, from Bacillus subtilis. Increased acetylation of TufA was observed during the exponential growth phase in LB and minimal glucose conditions, and its acetylation level decreased upon entering the stationary phase, while its succinylation increased during the late stationary phase. TufA was also succinylated during vegetative growth under minimal citrate or succinate conditions. Mutational analysis showed that triple succinylation mimic mutations at Lys306, Lys308 and Lys316 in domain-3 of TufA had a negative effect on B. subtilis growth, whereas the non-acylation mimic mutations at these three lysine residues did not. Consistent with the growth phenotypes, the triple succinylation mimic mutant showed 67 % decreased translation activity in vitro, suggesting a possibility that succinylation at the lysine residues in domain-3 decreases the translation activity. TufA, including Lys308, was non-enzymatically succinylated by physiological concentrations of succinyl-CoA. Lys42 in the G-domain was identified as the most frequently modified acetylation site, though its acetylation was likely dispensable for TufA translation activity and growth. Determination of the intracellular levels of acetylating substrates and TufA acetylation revealed that acetyl phosphate was responsible for acetylation at several lysine sites of TufA, but not for Lys42 acetylation. It was speculated that acetyl-CoA was likely responsible for Lys42 acetylation, though AcuA acetyltransferase was not involved. Zn2+-dependent AcuC and NAD+-dependent SrtN deacetylases were responsible for deacetylation of TufA, including Lys42. These findings suggest the potential regulatory roles of acetylation and succinylation in controlling TufA function and translation in response to nutrient environments in B. subtilis.

    • A genetic screen for mutations affecting temperature sensing in Bacillus subtilis

      Two component systems, composed of a receptor histidine kinase and a cytoplasmic response regulator, regulate pivotal cellular processes in microorganisms. Here we describe a new screening procedure for the identification of amino acids that are crucial for the functioning of DesK, a prototypic thermosensor histidine kinase from Bacillus subtilis. This experimental strategy involves random mutagenesis of the membrane sensor domain of the DesK coding sequence, followed by the use of a detection procedure based on changes in the colony morphogenesis that take place during the sporulation programme of B. subtilis. This method permitted us the recovery of mutants defective in DesK temperature sensing. This screening approach could be applied to all histidine kinases of B. subtilis and also to kinases of other bacteria that are functionally expressed in this organism. Moreover, this reporter assay could be expanded to develop reporter assays for a variety of transcriptionally regulated systems.

    • Post-transcriptional regulation of cholera toxin production in Vibrio cholerae by the stringent response regulator DksA

      Expression of cholera toxin (CT), the principal virulence factor of the cholera pathogen Vibrio cholerae, is positively modulated by the RNA polymerase binding unusual transcription factor DksA (DksAVc) of the stringent response pathway. Here we report that even though CT (encoded by the genes ctxAB) production is downregulated in the V. cholerae ΔdksA (ΔdksAVc ) mutant, the expression of the ctxA gene as well as the genes encoding different virulence regulators, namely, AphA, TcpP and ToxT, were also upregulated. Since DksAVc positively regulates HapR, a known negative regulator of CT production, the increased expression of different virulence genes in ΔdksAVc was due most probably to downregulation of HapR. There was no secretion/transport-related defect in ΔdksAVc cells because whole cell lysates of the mutant showed a negligible amount of CT accumulation similar to WT cells. To understand further, the hapR gene was deleted in ΔdksAVc background, however, the double mutant failed to rescue the CT production defect suggesting strongly towards post-transcriptional/translational regulation by DksAVc. This hypothesis was further confirmed when the site-directed mutagenesis of each or both of the conserved aspartic acid residues at positions 68 and 71 of DksAVc, which are essential for transcription initiation during the stringent response, had no effect in the regulation of CT expression. Interestingly, progressive deletion analysis indicated that the C4-type Zn finger motif present in the C-terminus of DksAVc is essential for optimal CT production. Since this motif plays important roles in DNA/RNA binding, the present study indicates a novel complex post-transcriptional regulation of CT expression by DksAVc.

    • Regulation of cid and lrg expression by CcpA in Streptococcus mutans

      The Streptococcus mutans Cid/Lrg system represents an ideal model for studying this organism’s ability to withstand various stressors encountered in the oral cavity. The lrg and cid operons display distinct and opposite patterns of expression in response to growth phase and glucose levels, suggesting that the activity and regulation of these proteins must be tightly coordinated in the cell and closely associated with metabolic pathways of the organism. Here, we demonstrate that expression of the cid and lrg operons is directly mediated by a global transcriptional regulator CcpA in response to glucose levels. Comparison of the cid and lrg promoter regions with the conserved CcpA binding motif revealed the presence of two potential cre sites (for CcpA binding) in the cid promoter (designated cid-cre1 and cid-cre2), which were arranged in a similar manner to those previously identified in the lrg promoter region (designated lrg-cre1 and lrg-cre2). We demonstrated that CcpA binds to both the cid and lrg promoters with a high affinity, but has an opposing glucose-dependent effect on the regulation of cid (positive) and lrg (negative) expression. DNase I footprinting analyses revealed potential binding sequences for CcpA in both cid and lrg promoter regions. Collectively, these data suggest that CcpA is a direct regulator of cid and lrg expression, and are suggestive of a potential mechanism by which Cid/Lrg-mediated virulence and cellular homeostasis is integrated with signals associated with both the environment and cellular metabolic status.

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