Acinetobacter baumannii is an opportunistic pathogen that causes nosocomial infections. Due to the ability to persist in the clinical environment and rapidly acquire antibiotic resistance, multidrug-resistant A. baumannii clones have spread in medical units in many countries in the last decade. The molecular basis of the emergence and spread of the successful multidrug-resistant A. baumannii clones is not understood. Bacterial toxin-antitoxin (TA) systems are abundant genetic loci harbored in low-copynumber plasmids and chromosomes and have been proposed to fulfill numerous functions, from plasmid stabilization to regulation of growth and death under stress conditions. In this study, we have performed a thorough bioinformatic search for type II TA systems in genomes of A. baumannii strains and estimated at least 15 possible TA gene pairs, 5 of which have been shown to be functional TA systems. Three of them were orthologs of bacterial and archaeal RelB/RelE, HicA/HicB, and HigB/HigA systems, and others were the unique SplT/SplA and CheT/CheA TA modules. The toxins of all five TA systems, when expressed in Escherichia coli, inhibited translation, causing RNA degradation. The HigB/HigA and SplT/SplA TA pairs of plasmid origin were highly prevalent in clinical multidrug-resistant A. baumannii isolates from Lithuanian hospitals belonging to the international clonal lineages known as European clone I (ECI) and ECII.

Enterobacteria have developed numerous constitutive and inducible strategies to sense and adapt to an external acidity. These molecular responses require dozens of specific acid shock proteins (ASPs), as shown by genomic and proteomic analysis. Most of the ASPs remain poorly characterized, and their role in the acid response and survival is unknown. We recently identified an Escherichia coli gene, asr (acid shock RNA), encoding a protein of unknown function, which is strongly induced by high environmental acidity (pH < 5.0). We show here that Asr is required for growth at moderate acidity (pH 4.5) as well as for the induction of acid tolerance at moderate acidity, as shown by its ability to survive subsequent transfer to extreme acidity (pH 2.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of acid-shocked E. coli cells harboring a plasmid-borne asr gene demonstrated that the Asr protein is synthesized as a precursor with an apparent molecular mass of 18 kDa. Mutational studies of the asr gene also demonstrated the Asr preprotein contains 102 amino acids. This protein is subjected to an N-terminal cleavage of the signal peptide and a second processing event, yielding 15-and 8-kDa products, respectively. Only the 8-kDa polypeptide was detected in acid-shocked cells containing only the chromosomal copy of the asr gene. N-terminal sequencing and site-directed mutagenesis revealed the two processing sites in the Asr protein precursor. Deletion of amino acids encompassing the processing site required for release of the 8-kDa protein resulted in an acid-sensitive phenotype similar to that observed for the asr null mutant, suggesting that the 8-kDa product plays an important role in the adaptation to acid shock. Analysis of Asr:PhoA fusions demonstrated a periplasmic location for the Asr protein after removal of the signal peptide. Homologues of the asr gene from other Enterobacteriaceae were cloned and shown to be induced in E. coli under acid shock conditions.

Soil is one of the biggest reservoirs of microbial diversity, yet the processes that define the community dynamics are not fully understood. Apart from soil management being vital for agricultural purposes, it is also considered a favorable environment for the evolution and development of antimicrobial resistance, which is due to its high complexity and ongoing competition between the microorganisms. Different approaches to agricultural production might have specific outcomes for soil microbial community composition and antibiotic resistance phenotype. Therefore in this study we aimed to compare the soil microbiota and its resistome in conventional and organic farming systems that are continually influenced by the different treatment (inorganic fertilizers and pesticides vs. organic manure and no chemical pest management). The comparison of the soil microbial communities revealed no major differences among the main phyla of bacteria between the two farming styles with similar soil structure and pH. Only small differences between the lower taxa could be observed indicating that the soil community is stable, with minor shifts in composition being able to handle the different styles of treatment and fertilization. It is still unclear what level of intensity can change microbial composition but current conventional farming in Central Europe demonstrates acceptable level of intensity for soil bacterial communities. When the resistome of the soils was assessed by screening the total soil DNA for clinically relevant and soil-derived antibiotic resistance genes, a low variety of resistance determinants was detected (resistance to β-lactams, aminoglycosides, tetracycline, erythromycin, and rifampicin) with no clear preference for the soil farming type. The same soil samples were also used to isolate antibiotic resistant cultivable bacteria, which were predominated by highly resistant isolates of Pseudomonas, Stenotrophomonas, Sphingobacterium and Chryseobacterium genera. The resistance of these isolates was largely dependent on the efflux mechanisms, the soil Pseudomonas spp. relying mostly on RND, while Stenotrophomonas spp. and Chryseobacterium spp. on RND and ABC transporters.

Bacterial toxin-antitoxin (TA) systems are operons that code for a stable toxic protein and a labile antitoxin. TA modules are widespread on the chromosomes of free-living Bacteria and Archaea, where they presumably act as stress response elements. The chromosome of Escherichia coli K-12 encodes four known TA pairs, as well as the dinJ-yafQ operon, which is hypothesized to be a TA module based on operon organization similar to known TA genes. Induction of YafQ inhibited cell growth, but its toxicity was counteracted by coexpression of dinJ cloned on a separate plasmid. YafQ(His)(6) and DinJ proteins coeluted in Ni(2+)-affinity and gel filtration chromatography, implying the formation of a specific and stable YafQ-DinJ protein complex with an estimated molecular mass of c. 37.3 kDa. Induction of YafQ reduced protein synthesis up to 40% as judged by incorporation of [(35)S]-methionine, but did not influence the rates of DNA and RNA synthesis. Structure modelling of E. coli YafQ revealed its structural relationship with bacterial toxins of known structure suggesting that it might act as a sequence-specific mRNA endoribonuclease.

Acinetobacter baumannii currently represents one of the most important nosocomial infection agent due to its multidrug-resistance and a propensity for the epidemic spread. The A. baumannii strains belonging to the international clonal lineages I (IC I) and II (IC II) are associated with the hospital outbreaks and a high virulence. However, the intra and inter lineage-specific features of strains belonging to these most worldwide spread A. baumannii clones are not thoroughly explored. In this study we have investigated a set of cell surface-related features of A. baumannii IC I (n = 20) and IC II (n = 16) lineage strains, representing 30 distinct pulsed-field gel electrophoresis types in the collection of clinical isolates obtained in Lithuanian tertiary care hospitals. We show that A. baumannii IC II strains are non-motile, do not form pellicle and display distinct capsular polysaccharide profile compared with the IC I strains. Moreover, in contrast to the overall highly hydrophobic IC I strains, IC II strains showed a greater variation in cell surface hydrophobicity. Within the IC II lineage, hydrophilic strains demonstrated reduced ability to form biofilm and adhere to the abiotic surfaces, also possessed twofold thicker cell wall and exhibited higher resistance to desiccation. Furthermore, these strains showed increased adherence to the lung epithelial cells and were more virulent in nematode and mouse infection model compared with the hydrophobic IC II strains. According to the polymerase chain reaction-based locus-typing, the reduction in hydrophobicity of IC II strains was not capsule or lipooligosaccharide locus type-dependent. Hence, this study shows that the most widespread A. baumannii clonal lineages I and II markedly differ in the series of cell surface-related phenotypes including the considerable phenotypic diversification of IC II strains at the intra-lineage level. These findings suggest that the genotypically related A. baumannii strains might evolve the features which could provide an advantage at the specific conditions outside or within the host.