- Volume 162, Issue 4, 2016

Mycobacterium avium subsp. paratuberculosis (MAP), the aetiological agent of Johne's disease, is one of the most important bacterial pathogens in ruminants. A thorough understanding of MAP pathogenesis is needed to develop new vaccines and diagnostic tests. The generation of comprehensive random transposon mutant libraries is a fundamental genetic technology to determine the role of genes in physiology and pathogenesis. In this study, whole MAP genome analysis compared the insertion sites for the mycobacterial transposon Tn5367 derived from the Mycobacterium smegmatis insertion sequence IS1096 and the mariner transposon MycoMarT7 carrying the Himar1 transposase. We determined that only MycoMarT7 provides a random representation of insertions in 99 % of all MAP genes. Analysis of the MAP K-10 genome indicated that 710 of all ORFs do not possess IS1096 recognition sites, while only 37 do not have the recognition site for MycoMarT7. Thus, a significant number of MAP genes remain underrepresented in insertion libraries from IS1096-derived transposons. Analysis of MycoMarT7 and Tn5367 mutants showed that Tn5367 has a predilection to insert within intergenic regions, suggesting that MycoMarT7 is the more adequate for generating a comprehensive library. However, we uncovered the novel finding that both transposons have loci-dependent biases, with Tn5367 being the most skewed. These loci-dependent transposition biases led to an underestimation of the number of independent mutants required to generate a comprehensive mutant library, leading to an overestimation of essential genes. Herein, we also demonstrated a useful platform for gene discovery and analysis by isolating three novel mutants for each transposon.

Streptococcus mutans, one of the primary causative agents of dental caries in humans, ferments dietary sugars in the mouth to produce organic acids. These acids lower local pH values, resulting in demineralization of the tooth enamel, leading to caries. To survive acidic environments, Strep. mutans employs several adaptive mechanisms, including a shift from saturated to unsaturated fatty acids in membrane phospholipids. PlsX is an acyl-ACP : phosphate transacylase that links the fatty acid synthase II (FASII) pathway to the phospholipid synthesis pathway, and is therefore central to the movement of unsaturated fatty acids into the membrane. Recently, we discovered that plsX is not essential in Strep. mutans. A plsX deletion mutant was not a fatty acid or phospholipid auxotroph. Gas chromatography of fatty acid methyl esters indicated that membrane fatty acid chain length in the plsX deletion strain differed from those detected in the parent strain, UA159. The deletion strain displayed a fatty acid shift similar to WT, but had a higher percentage of unsaturated fatty acids at low pH. The deletion strain survived significantly longer than the parent strain when cultures were subjected to an acid challenge of pH 2.5.The ΔplsX strain also exhibited elevated F-ATPase activity at pH 5.2, compared with the parent. These results indicate that the loss of plsX affects both the fatty acid synthesis pathway and the acid-adaptive response of Strep. mutans.

A Myxococcus xanthus gene, MXAN3487, was identified by transposon mutagenesis to be required for the expression of mcuABC, an operon coding for part of the chaperone–usher (CU) system in this bacterium. The MXAN3487 protein displays sequence and structural homology to adenosine 5′-phosphosulphate (APS) kinase family members and contains putative motifs for ATP and APS binding. Although the MXAN3487 locus is not linked to other sulphate assimilation genes, its protein product may have APS kinase activity in vivo and the importance of the ATP-binding site for activity was demonstrated. Expression of MXAN3487 was not affected by sulphate availability, suggesting that MXAN3487 may not function in a reductive sulphate assimilation pathway. Deletion of MXAN3487 significantly delayed fruiting body formation and the production of McuA, a spore coat protein secreted by the M. xanthus Mcu CU system. Based on these observations and data from our previous studies, we propose that MXAN3487 may phosphorylate molecules structurally related to APS, generating metabolites necessary for M. xanthus development, and that MXAN3487 exerts a positive effect on the mcuABC operon whose expression is morphogenesis dependent.

Desulfosporosinus sp. OT is a Gram-positive, acidophilic sulfate-reducing firmicute isolated from copper tailings sediment in the Norilsk mining-smelting area in Siberia and represents the first Desulfosporosinus species whose genome has been sequenced. Desulfosporosinus sp. OT is exceptionally copper resistant, which made it of interest to study the resistance mechanism. It possesses a copUAZ operon which is shown here to be involved in copper resistance. The copU gene encodes a CsoR-type homotetrameric repressor. By electrophoretic mobility shift assay, it was shown that CopU binds to the operator/promoter region of the copUAZ operon in the absence of copper and is released from the DNA by Cu+ or Ag+, implying that CopU regulates the operon in a copper/silver-dependent manner. DOT_CopA is a P1B-type ATPase related to other characterized, bacterial copper ATPases. When expressed in a copper-sensitive Escherichia coli ΔcopA mutant, it restores copper resistance to WT levels. His-tagged DOT_CopA was expressed from a plasmid in E. coli and purified by Ni-NTA affinity chromatography. The purified enzyme was most active in the presence of Cu(I) and bacterial phospholipids. These findings indicate that the copUAZ operon confers copper resistance to Desulfosporosinus sp. OT, but do not per se explain the basis of the high copper resistance of this strain.

Pyridoxal 5′-phosphate (PLP) is an essential cofactor for nearly 60 Escherichia coli enzymes but is a highly reactive molecule that is toxic in its free form. How PLP levels are regulated and how PLP is delivered to target enzymes are still open questions. The COG0325 protein family belongs to the fold-type III class of PLP enzymes and binds PLP but has no known biochemical activity although it occurs in all kingdoms of life. Various pleiotropic phenotypes of the E. coli COG0325 (yggS) mutant have been reported, some of which were reproduced and extended in this study. Comparative genomic, genetic and metabolic analyses suggest that these phenotypes reflect an imbalance in PLP homeostasis. The E. coli yggS mutant accumulates the PLP precursor pyridoxine 5′-phosphate (PNP) and is sensitive to an excess of pyridoxine but not of pyridoxal. The pyridoxine toxicity phenotype is complemented by the expression of eukaryotic yggS orthologs. It is also suppressed by the presence of amino acids, specifically isoleucine, threonine and leucine, suggesting the PLP-dependent enzyme transaminase B (IlvE) is affected. These genetic results lay a foundation for future biochemical studies of the role of COG0325 proteins in PLP homeostasis.

The lux-operon of the psychrophilic bioluminescent bacterium Aliivibrio logei is regulated by quorum sensing (QS). The key components of this system are LuxI, which catalyses synthesis of the autoinducer (AI), and LuxR, which activates transcription of the entire lux-operon. The lux-operon of A. logei contains two copies of the luxR gene: luxR1 and luxR2. In the present study, lux-operon sequence analysis from 16 strains of A. logei, isolated from cold habitats of the White, Baltic, Okhotsk and Bering seas, was carried out. Phylogenetic analysis showed that all isolated strains of A. logei have both copies of luxR genes which are homologous to luxR genes of the related Aliivibrio salmonicida. Evaluation of LuxR1 and LuxR2 activity showed that LuxR2 remains active at significantly lower concentrations of AI (10− 9 M) than LuxR1, which is active only at high AI concentrations (10− 6 M). As the QS response is already prominent at AI concentrations as low as 10− 8 to 10− 9 M, we conclude that LuxR2 is the main activator of the lux-operon of A. logei. The thermolabilities of LuxR1 and LuxR2 are similar and exceed that of LuxR of the mesophilic bacterium Aliivibrio fischeri. In contrast to LuxR2, LuxR1 is not a substrate of Lon protease and does not require the chaperonin GroEL/ES for its folding. This study expands our current understanding of QS regulation in A. logei as it implies differential regulation by LuxR1 and LuxR2 proteins.