- Volume 164, Issue 12, 2018

The principal mechanism of reducing sulfur into organic compounds is via the synthesis of l-cysteine. Cysteine is used for protein and glutathione synthesis, as well as being the primary sulfur source for a variety of other molecules, such as biotin, coenzyme A, lipoic acid and more. Glutathione and other cysteine derivatives are important for protection against the oxidative stress that pathogenic bacteria such as Neisseria gonorrhoeae and Neisseria meningitidis encounter during infection. With the alarming rise of antibiotic-resistant strains of N. gonorrhoeae, the development of inhibitors for the future treatment of this disease is critical, and targeting cysteine biosynthesis enzymes could be a promising approach for this. Little is known about the transport of sulfate and thiosulfate and subsequent sulfate reduction and incorporation into cysteine in Neisseria species. In this review we investigate cysteine biosynthesis within Neisseria species and examine the differences between species and with other bacteria. Neisseria species exhibit different arrangements of cysteine biosynthesis genes and have slight differences in how they assimilate sulfate and synthesize cysteine, while, most interestingly, N. gonorrhoeae by virtue of a genome deletion, lacks the ability to reduce sulfate to bisulfide for incorporation into cysteine, and as such uses the thiosulfate uptake pathway for the synthesis of cysteine.

Bifidobacterium longum DJO10A was previously demonstrated to be able to produce a broad-spectrum lantibiotic, but production in media was very limited and only periodically on solid media. Given the difficulty of obtaining these lantibiotic peptides using B. longum DJO10A due to its tightly controlled production, genes predicted to be required for its production and immunity were designed and codon optimized according to the preferred codon used by Lactococcus lactis. Since the lanR1 gene within this lantibiotic gene cluster was the only one without a characterized analogue from other lantibiotic gene clusters, its annotation was re-examined as it was previously suggested to be a regulatory protein. Lack of DNA binding motifs did not support this, and one current analysis suggested a high likelihood of it interacting with LanD. Therefore, gene lanR1 together with lanADMIT were codon optimized and synthesized. Those genes were then cloned into an efficient dual-plasmid nisin-controlled expression system in L. lactis. The addition of the lanR1 gene exhibited toxicity in E. coli, specifically causing a shorter cell size as observed by SEM. No toxicity was observed in L. lactis. While this production system did not result in the production of a bioactive lantibiotic by L. lactis, it did successfully produce all the peptides and enzymes encoded by the original lantibiotic gene cluster from B. longum, as confirmed by LC-MS. This will now facilitate efforts into determining the proper conditions required for these enzymes to produce a bioactive lantibiotic.

Micro-organisms often face multiple stresses in natural habitats. Individual stresses are well known to influence mutation rates and the spectra of mutational types, but the extent to which multiple stresses affect the genetic variation in populations is unknown. Here we investigate pair-wise combinations of nutritional stresses in Escherichia coli to determine their effect on mutation rates and mutational types. Environmental interactions modified both the rate and spectrum of mutations in double-limited environments, but the effects were not additive or synergistic relative to single stresses. Generally, bacteria in the mixed environments behaved as if one of the two single-stress stimuli was more dominant and the genetic variation seen with every dual limitation was intermediate between known patterns with individual stresses. The composition of mutational types with double stresses was also intermediate between individual stress patterns. At least with mutations, the single stressor results available are reasonable indicators of stress-induced genetic variation in multifaceted natural habitats. With the influence of 11 conditions available on mutational patterns, we can now also see the clustering of mutational types as a function of these environments.

M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Algorithm (AMyGDA), that measures how much M. tuberculosis is growing in each well of a 96-well microtitre plate. The plate used here has serial dilutions of 14 anti-tuberculosis drugs, thereby permitting the MICs to be elucidated. The three participating laboratories each inoculated 38 96-well plates with 15 known M. tuberculosis strains (including the standard H37Rv reference strain) and, after 2 weeks' incubation, measured the MICs for all 14 drugs on each plate and took a photograph. By analysing the images, we demonstrate that AMyGDA is reproducible, and that the MICs measured are comparable to those measured by a laboratory scientist. The AMyGDA software will be used by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to measure the drug susceptibility profile of a large number (>30000) of samples of M. tuberculosis from patients over the next few years.

Here we assessed the time course of rhizoplane colonization by the endophytic insect pathogenic fungus Metarhizium robertsii. We describe a method of quantifying root colonization of bean plants by M. robertsii using quantitative polymerase chain reaction (qPCR). Results of this method were compared to the standard plate count method using colony-forming units (c.f.u.). Both the c.f.u. and qPCR methods were used to monitor the time-course of haricot bean (Phaseolus vulgaris) colonization by a strain of M. robertsii that expresses the green fluorescent protein (ARSEF 2575-GFP) for colony verification. There was a strong correlation between the results of the c.f.u. and qPCR methods, indicating that both methods are well suited for the determination of colonization of P. vulgaris roots by M. robertsii. Primers for a catalase gene (cat) amplified DNA from M. robertsii, M. brunneum and M. guizhouense. Primers for a nitrogen response-regulator (nrr) additionally detected M. acridum and M. flavoviride, whereas Metarhizium perilipin-like protein (mpl) primers were specific to M. robertsii alone. However, cat was the only target that specifically amplified Metarhizium in experiments utilizing non-sterile soil. Endophytic colonization of P. vulgaris at 60 days post-inoculation with M. robertsii was detected from surface-sterilized roots with more sensitivity using our qPCR technique over the c.f.u. method. Our results suggest that there is a prolonged period of rhizoplane colonization by Metarhizium with transient, low-level endophytic colonization of the root system of P. vulgaris that persists for the entirety of the plant life cycle.

Directing the flow of protein traffic is a critical task faced by all cellular organisms. In Gram-negative bacteria, this traffic includes lipoproteins. Lipoproteins are synthesized as precursors in the cytoplasm and receive their acyl modifications upon export across the inner membrane. The third and final acyl chain is added by Lnt, which until recently was thought to be essential in all Gram-negatives. In this report, we show that Acinetobacter species can also tolerate a complete loss-of-function mutation in lnt. Absence of a fully functional Lnt impairs modification of lipoproteins, increases outer membrane permeability and susceptibility to antibiotics, and alters normal cellular morphology. In addition, we show that loss of lnt triggers a global transcriptional response to this added cellular stress. Taken together, our findings provide new insights on and support the growing revisions to the Gram-negative lipoprotein biogenesis paradigm.