- Volume 167, Issue 3, 2021

Several genome engineering methods have been developed for Rhodococcus . However, they suffer from limitations such as extensive cloning, multiple steps, successful expression of heterologous genes via plasmid etc. Here, we report a rapid method for performing genomic deletions/disruptions in Rhodococcus spp. using heterologous linear DNA. The method is cost effective and less labour intensive. The applicability of the method was demonstrated by successful disruption of rodA and orphan parA. None of the disrupted genes were found to be essential for the viability of the cell. Disruption of orphan parA and rodA resulted in elongated cells and short rods, respectively. This is the first report demonstrating disruption of rodA and orphan parA genes by electroporation of heterologous linear DNA in Rhodococcus spp.

The type VII protein secretion system (T7SS) has been characterized in members of the phyla Actinobacteria and Firmicutes. In mycobacteria the T7SS is intimately linked with pathogenesis and intracellular survival, while in Firmicutes there is mounting evidence that the system plays a key role in interbacterial competition. A conserved membrane-bound ATPase protein, termed EssC in   Staphylococcus aureus  , is a critical component of the T7SS and is the primary receptor for substrate proteins. Genetic diversity in the essC gene of   S. aureus   has previously been reported, resulting in four protein variants that are linked to specific subsets of substrates. Here we have analysed the genetic diversity of the T7SS-encoding genes and substrate proteins across   Listeria monocytogenes   genome sequences. We find that there are seven EssC variants across the species that differ in their C-terminal region; each variant is correlated with a distinct subset of genes for likely substrate and accessory proteins. EssC1 is most common and is exclusively linked with polymorphic toxins harbouring a YeeF domain, whereas EssC5, EssC6 and EssC7 variants all code for an LXG domain protein adjacent to essC. Some essC1 variant strains encode an additional, truncated essC at their T7 gene cluster. The truncated EssC, comprising only the C-terminal half of the protein, matches the sequence of either EssC2, EssC3 or EssC4. In each case the truncated gene directly precedes a cluster of substrate/accessory protein genes acquired from the corresponding strain. Across   L. monocytogenes   strains we identified 40 LXG domain proteins, most of which are encoded at conserved genomic loci. These loci also harbour genes encoding immunity proteins and sometimes additional toxin fragments. Collectively our findings strongly suggest that the T7SS plays an important role in bacterial antagonism in this species.

The present study was done to explore the diversity of lactic acid bacteria (LAB) associated with the gastrointestinal tract (GIT) of honeybee species endemic to northeastern Pakistan. Healthy worker bees belonging to Apis mellifera, A. dorsata, A. cerana and A. florea were collected from hives and the surroundings of a major apiary in the region. The 16S rRNA amplicon sequencing revealed a microbial community in A. florea that was distinct from the others in having an abundance of Lactobacillus and Bifidobacteria. However, this was not reflected in the culturable bacteria obtained from these species. The isolates were characterized for safety parameters, and 20 LAB strains deemed safe were evaluated for resistance to human GIT stresses like acid and bile, adhesion and adhesiveness, and anti-pathogenicity. The five most robust strains, Enterococcus saigonensis NPL780a, Lactobacillus rapi NPL782a, Lactobacillus kunkeei NPL783a, and NPL784, and Lactobacillus paracasei NPL783b, were identified through normalized Pearson (n) principal components analysis (PCA). These strains were checked for inhibition of human pathogens, antibiotic resistance, osmotic tolerance, metabolic and enzymatic functions, and carbohydrate utilization, along with antioxidative and cholesterol-removing potential. The findings suggest at least three strains (NPL 783a, 784 and 782a) as candidates for further in vitro and in vivo investigations of their potential health benefits and application as novel probiotic adjuncts.

l-Arabinose, a major constituent pentose of plant cell-wall polysaccharides, has been suggested to be a less preferred carbon source for fungi but to be a potential signalling molecule that can cause distinct genome-wide transcriptional changes in fungal cells. Here, we explore the possibility that this unique pentose influences the morphological characteristics of the phytopathogenic fungus Bipolaris maydis strain HITO7711. When grown on plate media under different sugar conditions, the mycelial dry weight of cultures on l-arabinose was as low as that with no sugar, suggesting that l-arabinose does not substantially contribute to vegetative growth. However, the intensity of conidiation on l-arabinose was comparable to or even higher than that on d-glucose and on d-xylose, in contrast to the poor conidiation under the no-sugar condition. To explore the physiological basis of the passive growth and active conidiation on l-arabinose, we next investigated cellular responses of the fungus to these sugar conditions. Transcriptional analysis of genes related to carbohydrate metabolism showed that l-arabinose stimulates carbohydrate utilization through the hexose monophosphate shunt (HMP shunt), a catabolic pathway parallel to glycolysis and which participates in the generation of the reducing agent NADPH (the reduced form of nicotinamide adenine dinucleotide phosphate). Then, the HMP shunt was impaired by disrupting the related gene BmZwf1, which encodes glucose-6-phosphate dehydrogenase in this fungus. The resulting mutants on l-arabinose showed remarkably decreased conidiation, but a conversely increased mycelial dry weight compared with the wild-type. Our study demonstrates that l-arabinose acts to enhance resource allocation to asexual reproduction in B. maydis HITO7711 at the cost of vegetative growth, and suggests that this is mediated by the concomitant stimulation of the HMP shunt.

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

Biofilm formation in the human intestinal pathogen Vibrio cholerae is in part regulated by norspermidine, spermidine and spermine. V. cholerae senses these polyamines through a signalling pathway consisting of the periplasmic protein, NspS, and the integral membrane c-di-GMP phosphodiesterase MbaA. NspS and MbaA belong to a proposed class of novel signalling systems composed of periplasmic ligand-binding proteins and membrane-bound c-di-GMP phosphodiesterases containing both GGDEF and EAL domains. In this signal transduction pathway, NspS is hypothesized to interact with MbaA in the periplasm to regulate its phosphodiesterase activity. Polyamine binding to NspS likely alters this interaction, leading to the activation or inhibition of biofilm formation depending on the polyamine. The purpose of this study was to determine the amino acids important for NspS function. We performed random mutagenesis of the nspS gene, identified mutant clones deficient in biofilm formation, determined their responsiveness to norspermidine and mapped the location of these residues onto NspS homology models. Single mutants clustered on two lobes of the NspS model, but the majority were found on a single lobe that appeared to be more mobile upon norspermidine binding. We also identified residues in the putative ligand-binding site that may be important for norspermidine binding and interactions with MbaA. Ultimately, our results provide new insights into this novel signalling pathway in V. cholerae and highlight differences between periplasmic binding proteins involved in transport versus signal transduction.

Rhodobacter sphaeroides can use C4-dicarboxylic acids to grow heterotrophically or photoheterotropically, and it was previously demonstrated in Rhodobacter capsulatus that the DctPQM transporter system is essential to support growth using these organic acids under heterotrophic but not under photoheterotrophic conditions. In this work we show that in R. sphaeroides this transporter system is essential for photoheterotrophic and heterotrophic growth, when C4-dicarboxylic acids are used as a carbon source. We also found that over-expression of dctPQM is detrimental for photoheterotrophic growth in the presence of succinic acid in the culture medium. In agreement with this, we observed a reduction of the dctPQM promoter activity in cells growing under these conditions, indicating that the amount of DctPQM needs to be reduced under photoheterotrophic growth. It has been reported that the two-component system DctS and DctR activates the expression of dctPQM. Our results demonstrate that in the absence of DctR, dctPQM is still expressed albeit at a low level. In this work, we have found that the periplasmic component of the transporter system, DctP, has a role in both transport and in signalling the DctS/DctR two-component system.