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From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of ~ 105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida. Upon screening two genomic libraries of A. pleuropneumoniae serotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp. HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box. The promoter region of hgbA from A. pleuropneumoniae serotype 1 showed consensus for -35 and -10 sequences and a putative Fur-binding site. RT-PCR confirmed that hgbA of A. pleuropneumoniae is upregulated in response to diminished levels of iron in the culture medium. While an internally deleted hgbA mutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb. HgbA is required for growth of A. pleuropneumoniae in the presence of Hb as sole iron source.

The mexR gene encoded product MexR in nalB mutants of Pseudomonas aeruginosa is modified by a single amino acid that compromizes the stability of the protein or its ability to dimerize. However, modification of L95 and R21 amino acid residues confer stability and dimerization capability but compromises DNA binding.

Results show that MexR, encoded by mexR repressor gene, functions solely as a repressor and controls the multidrug expression system and 5' end of the mexR gene may be the promoter of this expression system.

Research was conducted to test whether the outer membrane OprM protein influences beta-lactam recognition and efflux in Pseudomonas aeruginosa. The oprM gene was cloned on a SphI fragment into a derivative called pXZL01 while the MexCD-OprM efflux system was constructed and analyzed for reconstitution of efflux activity. Results showed that the efflux components are highly homologous and are characterized by an integral cytoplasmic membrane component, a linker protein and an outer membrane protein.

The porin protein of Haemophilus influenzae type b was analyzed using monoclonal antibodies (Mabs) to define the epitopic regions of the outer membrane protein. The results showed that the antigenic determinants of porin are located between amino acids 162 and 172 and between 318 and 325. Based on these results, as well as on the predicted secondary structure of porin, a model for the surface topological structure of porin is presented.

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Over evolutionary time bacteriophages have developed unique proteins that arrest critical cellular processes to commit bacterial host metabolism to phage reproduction. Here, we apply this concept of phage-mediated bacterial growth inhibition to antibiotic discovery. We sequenced 26 Staphylococcus aureus phages and identified 31 novel polypeptide families that inhibited growth upon expression in S. aureus. The cellular targets for some of these polypeptides were identified and several were shown to be essential components of the host DNA replication and transcription machineries. The interaction between a prototypic pair, ORF104 of phage 77 and DnaI, the putative helicase loader of S. aureus, was then used to screen for small molecule inhibitors. Several compounds were subsequently found to inhibit both bacterial growth and DNA synthesis. Our results suggest that mimicking the growth-inhibitory effect of phage polypeptides by a chemical compound, coupled with the plethora of phages on earth, will yield new antibiotics to combat infectious diseases. [ABSTRACT FROM AUTHOR]

Deletion of the mexAB-oprM multidrug efflux operon substantially compromised the β-lactam resistance of β-lactamase-derepressed mutants of Pseudomonas aeruginosa, although it had only a modest impact on resistance of a penicillin-binding protein mutant. This highlights the multifactorial nature of β-lactam resistance in this organism. Moreover, the contribution of efflux to the net resistance seen in some β-lactam-resistant mutants suggests that inhibition of MexAB-OprM-mediated drug efflux might be an effective approach to overcoming β-lactam resistance attributed to efflux as well as to other mechanisms of β-lactam resistance. [ABSTRACT FROM PUBLISHER]